Multifunctional alleles

ABSTRACT

Nucleic acid constructs and methods for rendering modifications to a genome are provided, wherein the modifications comprise null alleles, conditional alleles and null alleles comprising COINs. Multifunctional alleles (MFA) are provided, as well as methods for making them, which afford the ability in a single targeting to introduce an allele that can be used to generate a null allele, a conditional allele, or an allele that is a null allele and that further includes a COIN. MFAs comprise pairs of cognate recombinase recognition sites, an actuating sequence and/or a drug selection cassette, and a nucleotide sequence of interest, and a COIN, wherein upon action of a recombinase a conditional allele with a COIN is formed. In a further embodiment, action of a second recombinase forms an allele that contains only a COIN in sense orientation. In a further embodiment, action by a third recombinase forms an allele that contains only the actuating sequence in sense orientation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a nonprovisional of, and claims the benefit under 35USC 119(e) of, U.S. Ser. No. 61/256,078 filed 29 Oct. 2009, whichapplication is hereby incorporated by reference.

FIELD OF INVENTION

The invention relates to nucleic acid constructs for modifying genomes,including knockout constructs and constructs for placing COINs in agenome. Genetically modified non-human animals are included, e.g.,genetically modified mice having genes or nucleic acid elements arrayedwith selected recombinase recognition sites that allow for deletion orinversion of the genes or nucleic acid elements to form null alleles,selectable alleles, reporting alleles, and/or conditional alleles innon-human animals, e.g., in mice and rats.

BACKGROUND

Typically, knockouts are made by homologously replacing a target genewith another sequence of choice, usually a reporter and a selectioncassette, where the latter is preferably flanked by site-specificrecombinase sites to empower removal of the selection cassette via theaction of the cognate site-specific recombinase. The selection cassettecan be subsequently removed either by treating cells with thecorresponding cognate recombinase or by breeding mouse progeny to a“deletor” strain. For example, in the case of floxed alleles (where thesequence of interest is flanked by loxP sites), the cognate recombinaseis Cre, and what remains in the genome is a single loxP site and thereporter.

A related strategy has been traditionally employed to generateconditional-null alleles. This involves flanking part of the gene ofinterest with site-specific recombinase recognition sites (such as loxfor Cre, and FRT for Flp) in a manner such that upon action of thecognate recombinase, the region flanked by the site-specific recombinaserecognition sites is deleted and the resulting allele is a null allele.

Although attempts have been made to incorporate both a null and aconditional functionality in one targeting vector and to accomplishbuilding the corresponding modified alleles in a single targeting step,the methods that have resulted from such attempts have several drawbacksand have had mixed success. These drawbacks include, for example, lackof true functionality (i.e., the null version is not a true null, theconditional allele is not a true conditional, lack of reporter function,etc.) or inability to realize a practical working allele with thedesired features.

Therefore, there is a need in the art for generating geneticallymodified organisms via targeting where the engineered loci aremultifunctional loci, for example, a true KO-first allele and then aconditional-null or other conditional-mutant allele.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates use of recombinase recognition sites tosimultaneously delete one element (U) and invert another (D).

FIG. 2 illustrates an embodiment of a Multifunctional Allele (MFA)allele, shown for a nucleotide sequence of interest (NSI), employing asplice acceptor with splice region and an actuating sequence followed bya polyadenylation (pA) signal, a drug selection cassette (DSC; in asuitable orientation of choice), a COIN, and five pairs of recombinaserecognition sites. R1/R1, R2/R2′, R3/R3′, R4/R4′, and R5/R5′ representcognate pairs of recombinase recognition sites.

FIG. 3 illustrates a conceptual rendering of recombinable units (definedby R1/R1′, R2/R2′, R3/R3′, R4/R4′, and R5/R5′) of an embodiment of anMFA allele that employs an actuating sequence (for simplicity the spliceacceptor and splice region preceding the sequence, and polyA signalfollowing the sequence are not shown), a DSC (in a suitable orientationof choice), an NSI, and a COIN.

FIG. 4 illustrates a particular embodiment of an MFA with specificrecombinase recognition sites for purposes of illustration (top), andgenerating a “cleaned-up” null allele comprising an actuating sequencethat comprises a LacZ sequence, and removing the DSC as well as a theNSI and the COIN element from the initial MFA embodiment using a singlerecombinase step. For simplicity, the splice acceptor and splice regionpreceding the LacZ sequence, and the polyA signal following the LacZsequence, are not shown.

FIG. 5 illustrates a particular embodiment of an MFA that generates aconditional allele that contains/incorporates a COIN from an MFA using asingle recombinase (here, a Flp recombinase first acting on FRT3 sitesof the allele embodiment). For simplicity, the splice acceptor andsplice region preceding the LacZ sequence, and the polyA signalfollowing the LacZ sequence, are not shown.

FIG. 6 illustrates an embodiment of an MFA that generates a conditionalnull allele that contains/incorporates a COIN from an MFA using a singlerecombinase (here, a Flp recombinase first acting on FRT sites of theallele embodiment). For simplicity, the splice acceptor and spliceregion preceding the LacZ sequence, and the polyA signal following theLacZ sequence, are not shown.

FIG. 7 illustrates an embodiment wherein, following recombinasetreatment (Flp exposure as shown in FIG. 5 or FIG. 6), the allele isexposed to a second recombinase (Cre), resulting in deletion of the NSIand placement of the COIN in sense orientation for transcription.

FIG. 8 illustrates an embodiment wherein, following recombinasetreatment (Flp exposure), the allele is exposed to a second recombinase,resulting in inversion of the COIN and the NSI due to (an alternative)placement of a recombinase recognition site for the second recombinaseat a position 5′ of the NSI.

FIG. 9 illustrates the exon-intron structure of the mouse Hprt1 gene inthe region of exons 2 to 4, adapted from the Ensembl mouse genome server(top panel—www.ensembl.org), and the region from exon 2 to exon 4 isexpanded in the ECR browser (http://ecrbrowser.dcode.org) to highlightregions of conservation. Exon 3 is highlighted by a dotted oval. Theblack vertical arrow indicates the point of insertion of the actuatingsequence and DSC, whereas the gray arrow indicates the point ofinsertion of the COIN element, all used to engineer the Hprt1^(MFA)allele. Note that none of the evolutionarily conserved intronicsequences flanking exon 3 are disrupted in the resulting allele. Thedotted parallelogram denotes the region that will become the NSI in theHprt1^(MFA) allele.

FIG. 10 illustrates an example of an MFA, specifically the MFA for theHprt1 gene. Exon 3 plus evolutionarily conserved intronic sequencesflanking exon 3 (as illustrated in FIG. 9) of Hprt1 become the NSI. Upontargeting, the NSI is placed into the antisense strand with respect tothe direction of transcription of the Hprt1 gene. An actuatingsequence—SA-IacZ-polyA—and a DSC are placed upstream of the NSI. Theactuating sequence is placed in the sense orientation with respect tothe direction of transcription of the Hprt1 gene, effectively acting asa gene-trap element, and abrogating transcription downstream of theactuating sequence. A COIN element is placed downstream of the NSI inthe antisense orientation with respect to the direction of transcriptionof the Hprt1 gene. Neither the COIN element nor the NSI can beincorporated into a productive Hprt1 mRNA and therefore the resultingallele, Hprt1^(MFA), is a null allele with a reporter (LacZ). Theelements comprising the Hprt1^(MFA) allele are flanked by site-specificrecombinase recognition sites arranged as follows: FRT-actuatingsequence—Rox-DSC-FRT3-(LoxP)-(NSI)-(Lox2372)-(FRT)-(FRT3)-(COIN)-(Lox2372)-(LoxP)-Rox,where parenthesis denotes placement in the antisense orientation withrespect to the direction of transcription of the Hprt1 gene, or in thecase of site-specific recombinase sites opposite orientation withrespect to mutually recognized pairs.

FIG. 11 illustrates an embodiment of an MFA, showing certain overlappingrecombinable units (A) and resulting alleles that are generated by theaction of a first recombinase (B) or a second (C) and third (D)recombinase.

FIG. 12 illustrates an embodiment of an MFA, showing certain overlappingrecombinable units (A) and resulting alleles that are generated by theaction of a first recombinase (B) or a second (C) and third (D)recombinase.

FIG. 13 illustrates an embodiment of an MFA (A), showing resultingalleles that result from action of a first recombinase that places theNSI in sense orientation (B) and a second recombinase that places theNSI in antisense orientation while placing the COIN in sense orientation(C).

FIG. 14 illustrates an embodiment of an MFA (A), showing resultingalleles that result from action of a first recombinase that places theNSI in sense orientation (B) and a second recombinase that places theNSI in antisense orientation while placing the COIN in sense orientation(C).

FIG. 15 illustrates another embodiment of an MFA (A), showing resultingalleles that result from action of a first recombinase that places theNSI in sense orientation (B) and a second recombinase that places theNSI in antisense orietnation while placing the COIN in sense orientation(C).

FIG. 16 illustrates an example of an MFA embodiment, wherien thereporter is a SA(adml)-gtx-LacZ-pA, the DSC is Neo, the NSI is acritical exon (e_(c)), and the COIN is Gtx-SA-HA-myc3-TM-T2A-GFP-pA (A),placement of the NSI in sense orientation by action of a recombinasewhile maintaining the COIN in antisense orientation (B), and furtherexcision of the NSI with concomitant placement of the COIN in senseorientation (C); arrows indicate primers used to confirm identities andorientations of recombinase sites in the MFA (A), and upon recombinasetreatment (B and C).

FIG. 17 shows the results of cell viability and proliferation assays forHprt1⁺/Y, Hprt1^(MFA)/Y, Hprt1^(COIN)/Y, and Hprt1^(COIN-INV)/Y cellsrespectively, all cultured in standard ES cell culture media eitherwithout 6-TG (no 6-TG; upper panels) or supplemented with 10 μM 6-TG(6-TG; lower panels).

FIG. 18 shows Western blots of total protein preparations derived fromHprt1⁺/Y cells (WT), Hprt1^(MFA)/Y (MFA)—i.e. cells targeted with theMFA of FIG. 16A, Hprt1^(COIN)/Y cells (MFA+FLPo)—i.e. cells targetedwith the MFA of FIG. 16A and then treated with FLPo, andHprt1^(COIN-INV)/Y cells (MFA+FLPo+Cre)—i.e. Hprt1^(COIN)/Y cellstreated with Cre; the top panel shows detection of Hprt1 protein, thecenter panel shows detection of LacZ (reporter) protein, and bottompanel shows detection of GAPDH protein as a loading control.

SUMMARY

Methods and compositions for making null alleles and conditionalalleles, and alleles that combine null and COIN features, are provided.In various embodiments, methods and compositions are provided forengineering multifunctional alleles into a genome in a single targetingstep. Methods and compositions for knockout complementation analysis ingenetically modified nonhuman animals are provided, including methodsthat comprise a single targeting step.

In one aspect, a modified allele is provided, comprising a 3′ spliceregion and splice acceptor, an actuating sequence 3′ with respect to thesplice acceptor, and a nucleotide sequence of interest (NSI) 3′ withrespect to the actuating sequence, wherein the NSI is in antisenseorientation with respect to the target gene (or the locus beingmodified, or with respect to the actuating sequence).

In one embodiment, the actuating sequence is selected from a microRNA, atranscriptional stop signal (such as a polyadenylation region), anucleotide sequence encoding a cDNA, or any combinations thereof, andmay include regulatory elements such as operators, enhancers, andinsulators. In a specific embodiment, the cDNA encodes a reporter (e.g.,encodes for LacZ). In one embodiment, the actuating sequence comprisesan exon. In a specific embodiment, the exon is the 5′-most exon of alocus.

In one embodiment, the NSI comprises an exon. In one embodiment, the NSIcomprises an exon and neighboring intronic sequence. In a specificembodiment, the flanking exon is flanked 5′ and 3′ with intronicsequence. In one embodiment, the nucleotide sequence comprises two ormore exons, and in a specific embodiment comprises intronic sequence(s).In another embodiment, the NSI lacks an exon, or lacks a fragment of anexon.

In one embodiment, the modified allele comprises a COIN. In oneembodiment, the COIN is 3′ with respect to the NSI; in anotherembodiment, the COIN is 5′ with respect to the NSI.

In one embodiment, the COIN is selected from a reporter, a genetrap-like element (GT-like element), and a gene trap-like reporter(GT-like reporter). In a specific embodiment, the GT-like element isselected from SA-drug resistance cDNA-polyA. In a specific embodiment,the GT-like reporter is selected from SA-reporter-polyA.

In one embodiment, the COIN comprises a 3′ splice region. In a specificembodiment, the 3′ splice region is followed by a sequence selected froma cDNA, an exon-intron sequence, a microRNA, a microRNA cluster, a smallRNA, a codon-skipping element, an IRES, a polyadenylation sequence, orany combination thereof. In a specific embodiment, the small RNA is amirtron. In a specific embodiment, the codon-skipping element is T2A,E2A, or F2A.

In one embodiment, the modified allele comprises a drug selectioncassette (DSC).

In one embodiment, the modified allele is on a targeting construct thatcomprises an upstream and a downstream homology arm. In one embodiment,at least one homology arm is a mouse homology arm. In a specificembodiment, both homology arms are mouse homology arms.

In one embodiment, the modified allele comprises, from 5′ to 3′, asplice acceptor, an actuating sequence, a DSC, a NSI and a COIN whereinthe nucleotide sequence of interest and the COIN are both in antisenseorientation with respect to the actuating sequence, and five pairs ofsite-specific recombinase recognition sites. In one embodiment, themodified allele, upon exposure to a first site-specific recombinase thatindependently recognizes and inverts sequence between a first pair ofthe site-specific recombinase recognition sites and deletes a sequencebetween a second pair of the site-specific recombinase recognitionsites, results in an allele that comprises the NSI in sense orientationfor transcription, that lacks the DSC, and that comprises the COIN inantisense orientation. In one embodiment, the modified allele comprisesthird and fourth site-specific recombinase recognition sites arrangedsuch that further exposure of the allele to a second recombinase thatindependently recongizes the third and fourth site-specific recombinaserecognition sites results in deleting the NSI and placing the COIN insense orientation for transcription.

In one aspect, a nucleic acid construct is provided that comprises (a) areporter in sense orientation and a DSC in a suitable orientation ofchoice, and in antisense orientation a NSI and a COIN; (b) five pairs ofsite specific recombinase recognition sites, wherein the five pairs ofrecombinase recognition sites are recognized by no more than threerecombinases; wherein upon treatment of the nucleic acid construct witha first recombinase, a modified allele is formed wherein (i) the NSI isplaced in sense orientation, (ii) the COIN remains in antisenseorientation, (iii) the reporter and the DSC are deleted, and, (iv) themodified allele upon treatment with a second recombinase inverts and/ordeletes the NSI and places the COIN in sense orientation.

In one embodiment, the five pairs of site-specific recombinaserecognition sites are FRT3, Rox, FRT, loxP, and lox2372 pairs.

In one embodiment, the first recombinase is a Flp recombinase, and thesecond recombinase is a Cre recombinase.

In one embodiment, the modified allele upon treatment with the secondrecombinase results in an allele that cannot be deleted or inverted bythe first or the second recombinase.

In one embodiment, the nucleotide sequence of interest is a wild-typeexon of a gene. In another embodiment, the NSI is an exon of a genehaving one or more nucleic acid substitutions, deletions, or additions.

In one embodiment, the NSI is a wild-type exon plus intronic flanking ofa gene. In another embodiment, the NSI is an exon plus neighboringintronic sequence of a gene having one or more nucleic acidsubstitutions, deletions, or additions.

In one embodiment, the NSI is a wild type intron of a gene. In anotherembodiment, the NSI is an intron of a gene having one or more nucleicacid substitutions, deletions, or additions.

In one embodiment, the COIN comprises an exon or exons of a gene thatcomprises one or more nucleic acid substitutions, deletions, oradditions. In a specific embodiment, the COIN comprises an exon of amammal. In a specific embodiment, the mammal is a human, mouse, monkey,or rat.

In one embodiment, the COIN comprises a 3′ splice region. In a specificembodiment, the 3′ splice region is followed by a sequence selected froma cDNA, an exon-intron sequence, a microRNA, a microRNA cluster, a smallRNA, a codon-skipping element, an IRES, a polyadenylation sequence, anda combination thereof. In a specific embodiment, the small RNA is amirtron. In a specific embodiment, the codon-skipping element is a T2A.

In one embodiment, the COIN is selected from a reporter, a genetrap-like element (GT-like element), and a gene trap-like reporter(GT-like reporter). In a specific embodiment, the GT-like element isselected from SA-drug resistance cDNA-polyA. In a specific embodiment,the GT-like reporter is selected from SA-reporter-polyA.

In one embodiment, the construct further comprises an upstream and adownstream homology arm. In one embodiment, the upstream and thedownstream homology arm are mouse or rat homology arms. In a specificembodiment, the homology arms are mouse homology arms and the NSIcomprises a human sequence. In a specific embodiment, the human sequencecomprises a human exon that is a human homolog of a mouse exon.

In one embodiment the reporter is selected from: a fluorescent protein,a luminescent protein, or an enzyme. In a specific embodiment, thereporter is selected from GFP, eGFP, CFP, YFP, eYFP, BFP, eBFP, DsRed,MmGFP, luciferase, LacZ, and alkaline phosphatase.

In one embodiment the DSC comprises a sequence that encodes an activityselected from neomycin phosphotransferase (neo^(r)), hygromycin Bphosphotransferase (hyg^(r)), puromycin-N-acetyltransferase (puro^(r)),blasticidin S deaminase (bsr^(r)), xanthine/guanine phosphoribosyltransferase (gpt), nourseothricin acetyltransferase (nat1), and Herpessimplex virus thymidine kinase (HSV-tk).

In one aspect, a nucleic acid construct is provided that comprises anactuating sequence that comprises a 3′ splice acceptor followed by areporter in sense orientation, DSC in a suitable orientation of choice,a NSI in antisense orientation, and a COIN in antisense orientation,wherein the actuating sequence and reporter is flanked upstream by arecombinase recognition site R1, a recombinase recognition site R2 isdisposed between the reporter and the DSC, a recombinase site R3 isdisposed between the DSC and the nucleotide sequence of interest, arecombinase site R4 is disposed between the site R3 and the NSI, arecombinase site R5 is disposed between the NSI and the COIN, arecombinase site R1′ is disposed between site R5 and the COIN, arecombinase site R3′ is disposed between R1′ and the COIN, arecombination site R5′ is disposed downstream of the COIN, arecombination site R4′ is disposed downstream of site R5′, and arecombination site R2′ is disposed downstream of site R4′; wherein R1and R1′ are in opposite orientation, R2 and R2′ are in the sameorientation, R3 and R3′ are in opposite orientation, R4 and R4′ are inthe same orientation, and R5 and R5′ are in the same orientation.

In one embodiment, the reporter is followed by a polyadenylation region.

In one embodiment, R1 and R1′ are recognized by a recombinase thatrecognizes R3 and R3′. In one embodiment, R4 and R4′ are recognized by arecombinase that recognizes R5 and R5′. In one embodiment, R2 and R2′are not recognized by any recombinase that recognizes R1/R1′, R3/R3′,R4/R4′, or R5/R5′. In one embodiment, R1 and R1′, R3 and R3′, and R2 andR2′ are not recognized by any recombinase that recognizes R4 and R4′,and R5 and R5′. In one embodiment, R4 and R4′, R5 and R5′, and R2 andR2′ are not recognized by any recombinase that recognizes R1 and R1′ andR3 and R3′.

In one embodiment, treatment with a single recombinase results in anucleic acid construct that lacks the DSC, the NSI, and the COIN. In aspecific embodiment, the resulting nucleic acid construct consistsessentially of the actuating sequence, R1, and R2 or R2′. In a specificembodiment, R1 is a FRT3 site and R2 (or R2′) is a Rox site.

In one embodiment, treatment with a single recombinase results in anucleic acid construct that comprises the actuating sequence in senseorientation but that lacks the DSC, lacks the NSI, and lacks the COIN.In a specific embodiment, R2 and R2′ are Rox sites, and the singlerecombinase is Dre recombinase.

In one embodiment, treatment with a single recombinase results in anucleic acid construct that comprises the NSI in the antisenseorientation and the COIN in antisense orientation. In one embodiment,the single recombinase is a Flp recombinase, R1 and R1′ are a FRTvariant sequence that does not cross-react with R3 and R3′ (which arealso FRT or FRT variants), R2 and R2′ are Rox sequences, and R4 and R4′are loxP or lox variant sequences that do not cross-react with R5 andR5′, wherein R5 and R5′ are lox variant sequences.

In one embodiment, treatment with a single recombinase results in anucleic acid construct that comprises the NSI in sense orientation andthe COIN in antisense orientation. In one embodiment the singlerecombinase is a Flp recombinase, R1 and R1′ are FRT3 sequences, R2 andR2′ are Rox sequences, R3 and R3′ are FRT sequences, R4 and R4′ are loxPsequences, R5 and R5′ are lox2372 sequences.

In one embodiment, the NSI is a wild type exon of a gene. In anotherembodiment, the NSI is an exon of a gene having one or more nucleic acidsubstitutions, deletions, or additions.

In one embodiment, the COIN comprises an exon or exons of a gene thatcomprises one or more nucleic acid substitutions, deletions, oradditions. In a specific embodiment, the COIN comprises an exon of amammal. In one embodiment, the mammal is a human, mouse, monkey, or rat.

In one embodiment, the construct further comprises a homology armupstream of the construct (an upstream homology arm) and a homology armdownstream of the construct (a downstream homology arm). In oneembodiment, the upstream and the downstream homology arm are mouse orrat homology arms. In a specific embodiment, the homology arms are mousehomology arms and the NSI comprises a human sequence. In a specificembodiment, the human sequence comprises a human exon homologous to amouse exon.

In one embodiment the reporter is selected from: a fluorescent protein,a luminescent protein, or an enzyme. In a specific embodiment, thereporter is selected from GFP, eGFP, CFP, YFP, eYFP, BFP, eBFP, DsRed,MmGFP, luciferase, LacZ, and alkaline phosphatase. In one embodiment theDSC comprises a sequence that encodes an activity selected from neomycinphosphotransferase (neo^(r)), hygromycin B phosphotransferase (hyg^(r)),puromycin-N-acetyltransferase (puro^(r)), blasticidin S deaminase(bsr^(r)), xanthine/guanine phosphoribosyl transferase (gpt),nourseothricin acetyltransferase (nat1), and Herpes simplex virusthymidine kinase (HSV-tk).

In one aspect, a nucleic acid construct for modifying a locus isprovided, comprising a first, second, third, fourth, and fifthoverlapping recombinable unit, wherein a recombinable unit includes apair of cognate site-specific recombinase recognition sites, and wherein(a) the first recombinable unit is framed by recombinase sites R1 andR1′ in opposition orientation (allowing inversion via R1/R1′), whereinbetween R1 and R1′ are disposed an actuating sequence in senseorientation with respect to direction of transcription of the targetgene followed by a recombinase site R2 followed by a DSC in a suitableorientation of choice followed by a recombinase site R3 followed by arecombinase site R4 followed by a NSI in antisense orientation followedby a recombinase site R5; (b) the second recombinable unit is framed byrecombinase sites R2 and R2′ in the same orientation (allowing deletionvia R2/R2′), wherein between R2 and R2′ are disposed a DSC in a suitableorientation of choice followed by R3 followed by R4 followed by the NSIin antisense orientation followed by R5 followed by R1′ followed byrecombinase site R3′ wherein R3′ is in opposition orientation withrespect to R3 (enabling inversion via R3/R3′), followed by a COIN inantisense orientation, followed by R5′ wherein R5′ is in the sameorientation with respect to R5 followed by R4′ wherein R4′ is in thesame orientation with respect to R4 followed by R2′ wherein R2′ is inthe same orientation with respect to R2 (allowing deletion via R2/R2′);(c) the third recombinable unit is framed by recombinase sites R3 andR3′ in opposite orientation (allowing inversion via R3/R3′), whereinbetween R3 and R3′ are disposed R4, the NSI in antisense orientationfollowed by R5 followed by R1′; (d) the fourth recombinable unit framedby recombinase sites R4 and R4′ in the same orientation, wherein betweenR4 and R4′ are disposed the NSI in antisense orientation followed by R5followed by R1′ followed by R3′ followed by the COIN in antisenseorientation followed by R5′ followed by R4′; and, (e) the fifthrecombinable unit is framed by R5 and R5′ in the same orientation,wherein between R5 and R5′ are disposed R1′ followed by R3′ followed bythe COIN in antisense orientation.

In one embodiment, R1/R1′ and R3/R3′ are functional with respect to thesame site-specific recombinase, and said same site-specific recombinaseis not functional with respect to R4/R4′ and R5/R5′, and R2/R2′.

In one embodiment, R4/R4′ and R5/R5′ are functional with respect to thesame site specific recombinase, and said same site-specific recombinaseis not functional with respect to R1/R1′ and R3/R3′, and R2/R2′.

In one embodiment, R2/R2′ are functional with a recombinase, whereinsaid recombinase is not functional with respect to any of R1/R1′,R3/R3′, R4/R4′, and R5/R5′.

In one embodiment R1/R1′ are FRT, FRT3, loxP, or lox2372 sites. In oneembodiment R3/R3′ are FRT, FRT3, loxP, or lox2372 sites. In oneembodiment R4/R4′ are FRT, FRT3, loxP, or lox2372 sites. In oneembodiment, R5/R5′ are FRT, FRT3, loxP, or lox2372 sites. In oneembodiment, R2/R2′ are Rox sites. In one embodiment, R2/R2′ areattPlattB sites.

In a specific embodiment, R1/R1′ and R3/R3′ are functional with a Flprecombinase. In another specific embodiment, R1/R1′ and R3/R3′ arefunctional with a Cre recombinase.

In a specific embodiment, R4/R4′ and R5/R5′ are functional with a Crerecombinase. In another specific embodiment, R4/R4′ and R5/R5′ arefunctional with a Flp recombinase.

In one embodiment, R2/R2′ are Rox sites that are functional with a Drerecombinase. In another embodiment, R2/R2′ are attP/attB sites that arefunctional with PhiC31 integrase (phiC31\int).

In one embodiment the reporter is selected from: a fluorescent protein,a luminescent protein, or an enzyme. In a specific embodiment, thereporter is selected from GFP, eGFP, CFP, YFP, eYFP, BFP, eBFP, DsRed,MmGFP, luciferase, LacZ, and Alkaline Phosphatase.

In one embodiment the DSC comprises a sequence that encodes an activityselected from neomycin phosphotransferase (neo^(r)), hygromycin Bphosphotransferase (hyg^(r)), puromycin-N-acetyltransferase (puro^(r)),blasticidin S deaminase (bsr^(r)), xanthine/guanine phosphoribosyltransferase (gpt), nourseothricin acetyltransferase (nat1), and Herpessimplex virus thymidine kinase (HSV-tk).

In one embodiment, the NSI is a wild-type exon of a gene. In anotherembodiment, the NSI is an exon of a gene having one or more nucleic acidsubstitutions, deletions, or additions.

In one embodiment, the COIN comprises an exon of a gene that comprisesone or more nucleic acid substitutions, deletions, or additions. In aspecific embodiment, the COIN comprises an exon of a human, mouse,monkey, or rat.

In one embodiment, the COIN comprises a 3′ splice region. In a specificembodiment, the 3′ splice region is followed by a sequence selected froma cDNA, an exon-intron sequence, a microRNA, a microRNA cluster, a smallRNA, a codon-skipping element, an IRES, a polyadenylation sequence, anda combination thereof. In a specific embodiment, the small RNA is amirtron. In a specific embodiment, the codon-skipping element is T2A,E2A, or F2A.

In one embodiment, the COIN is selected from a reporter, a genetrap-like element (GT-like element), and a gene trap-like reporter(GT-like reporter). In a specific embodiment, the GT-like element isselected from SA-drug resistance cDNA-polyA. In a specific embodiment,the GT-like reporter is selected from SA-reporter-polyA.

In one embodiment, the construct further comprises a homology armupstream of the construct (an upstream homology arm) and a homology armdownstream of the construct (a downstream homology arm). In oneembodiment, the upstream and the downstream homology arm are mouse orrat homology arms. In a specific embodiment, the homology arms are mousehomology arms and the NSI comprises a human sequence. In a specificembodiment, the human sequence comprises a human exon homologous to amouse exon.

In one aspect, a multifunctional allele is provided comprising two ormore recombinable units that are recognized by two or more differentrecombinases, each recombinable unit defined by a pair of compatiblerecombinase recognition sites that define the boundaries of therecombinable unit. Each recombinable unit comprises one or more internalrecombinase recognition sites. The one or more internal recombinaserecognition sites are selected such that, upon recombination by a firstrecombinase of a recombinable unit of the multifunctional allele, theone or more internal recombinase recognition sites within onerecombinable unit then pair with one or more internal recombinase unitswithin another recombinable unit to allow for the inversion and/ordeletion by the first recombinase of a sequence that straddles two ormore recombinable units of the multifunctional allele, wherein theinversion and/or deletion is possible only upon inversion of the one ormore internal recombinase recognition sites.

In one embodiment, the inversion and/or deletion is accompanied byinversion of a further recombinase recognition site of themultifunctional allele, wherein inversion of the further recombinaserecognition site allows for the inversion or deletion of an element ofthe multifunctional allele by a second recombinase.

In one aspect, a multifunctional allele is provided, comprising: (a) afirst, a second, a third, a fourth, and a fifth recombinable unit,wherein each recombinable unit is bounded by compatible recombinaserecognition sites and wherein the first recombinable unit overlaps thesecond recombinable unit, and wherein the third, fourth, and fifthrecombinable units are contained within the second recombinable unit;(b) a first recombinable unit comprising a 3′ splice acceptor and spliceregion operably linked to an actuating sequence, a DSC, and a NSI; (c) asecond recombinable unit comprising the DSC, the NSI, and a COIN; (d) athird recombinable unit comprising the NSI; (e) a fourth recombinableunit comprising the NSI and the COIN; (f) a fifth recombinable unitcomprising the COIN; wherein multifunctional alleles comprise a firstpair of recombinase recognition sites flanking the first recombinableunit upstream and downstream that allow in a first inversion of thefirst recombinable unit, wherein the first inversion results in a secondinversion of a recombinase site within the second recombinable unit,wherein the second inversion orients the recombinase site within thesecond recombinable unit so as to delete the actuating sequence anddelete the DSC.

In one embodiment, a single recombinase recognizes the first pair ofrecombinase recognition sites and also deletes the actuating sequenceand the drug selection cassette.

In one embodiment, the second inversion orients a recombination sitesuch that following the inversion a second set of recombinaserecognition sites are formed that allow deletion of the NSI and/orinversion of the COIN.

In one aspect, a nucleic acid construct is provided, comprising a MFAcomprising, from 5′ to 3′ with respect to the direction oftranscription, a COIN in antisense orientation, a NSI in antisenseorientation, a DSC, and a reporter in sense orientation, wherein upontreatment of the MFA with a selected recombinase, the COIN, the NSI, andthe DSC are excised and the reporter remains in sense orientation; andwherein upon an alternate treatment with a different selectedrecombinase, the reporter and the DSC are excised, the COIN remains inantisense orientation, and the NSI is placed in sense orientation, suchthat upon a further treatment with yet another different selectedrecombinase, the NSI is excised and the COIN is placed in senseorientation.

In one embodiment, the MFA comprises a first recombinable unit, a secondrecombinable unit, and a third recombinable unit, wherein the firstrecombinable unit overlaps the second and third recombinable units, andwherein the second recombinable unit overlaps the first and thirdrecombinable units.

In one embodiment, the first recombinable unit comprises a COIN ininverse (antisense) orientation and an NSI in inverse orientation,wherein the recombinable unit is flanked upstream of the COIN anddownstream of the NSI by compatible recombinase sites R2 and R2′oriented to direct a deletion; the second recombinable unit overlaps thefirst recombinable unit, and the second recombinable unit isrecombinable by action of a recombinase on a recombination site upstreamof the DSC and a recombination site downstream of the reporter, whereinthe recombination sites are oriented to direct an inversion, and whereinthe recombination site upstream of the DSC is followed by a sequencecomprising the NSI. In a specific embodiment, the MFA comprises, from 5′to 3′ with respect to orientation on a sense strand, a first recombinasesite R1, a second recombinase site R2, a third recombinase site R3, theCOIN in antisense orientation, a fourth recombinase site R4, a fifthrecombinase site R5, a sixth recombinase site R3′ that is compatiblewith R3 and oriented to direct a deletion of sequence between R3 andR3′, the NSI in antisense orientation, a seventh recombinase site R2′that is compatible with R2 and oriented to direct a deletion of sequencebetween R2 and R2′, an eight recombinase site R4′, a DSC, a ninthrecombinase site R1′ that is compatible with R1 and oriented to direct adeletion of sequence between R1 and R1, a reporter in sense orientation,and a tenth recombinase site R5′ that is compatible with R5 and orientedto direct an inversion of sequence between R5 and R5′.

In a specific embodiment, R1/R1′ are Rox sites, R2/R2′ are loxP sites,R3/R3′ are lox 2372 sites, R4/R4′ are FRT sites, and R5/R5′ are FRT3sites. In a specific embodiment, the MFA comprises a placement ofrecombinase sites and COIN, NSI, DSC, and reporter as shown in FIG. 11,Panel A. In a specific embodiment, upon exposure to a single recombinasethat recognizes R1/R1′, an allele as shown in FIG. 11, Panel B isformed. In a specific embodiment, upon exposure to a single recombinasethat recognizes R4/R4′ and R5/R5′, an allele as shown in FIG. 11, PanelC is formed. In a specific embodiment, upon exposure of the allele ofFIG. 11, Panel C to a further recombinase that recognizes R2/R2′ andR3/R3′, an allele as shown in FIG. 11, Panel D is formed.

In one aspect, a nucleic acid construct is provided, comprising a MFAcomprising, from 5′ to 3′ with respect to the direction oftranscription, a NSI in antisense orientation, a DSC, a reporter insense orientation, and a COIN in antisense orientation; wherein upontreatment of the MFA with a selected recombinase, the NSI and the DSCare excised, the reporter remains in sense orientation, and the COINremains in antisense orientation; and wherein upon an alternatetreatment with a different selected recombinase, the DSC and thereporter are excised, and the NSI is placed in sense orientation and theCOIN is in antisense orientation, and wherein following the alternatetreatment with the different selected recombinase, the allele is treatedwith yet another different selected recombinase resulting in NSIexcision and placement of the COIN in sense orientation.

In one embodiment, the MFA comprises a first recombinable unit, a secondrecombinable unit, and a third recombinable unit, wherein the firstrecombinable unit overlaps the second and third recombinable units, andwherein the second recombinable unit overlaps the first and thirdrecombinable units. In one embodiment, the first recombinable unitcomprises a DSC and a reporter in sense orientation, wherein therecombinable unit is flanked upstream of the DSC by recombination sitesR2 followed by R3, and flanked downstream of the reporter by recombinasesite R3′ wherein R2/R3′ are oriented to direct an inversion, and whereinthe DSC is preceded by R2′ oriented with respect to R2 to direct aninversion; the second recombinable unit is flanked upstream of theantisense NSI by R4 and flanked downstream of the antisense COIN by R4′wherein R4/R4′ are oriented to direct an excision, and wherein thesecond recombinable unit includes the DSC and reporter; and the thirdrecombinable unit is flanked upstream by R1 and downstream by R1′,wherein R1/R1′ are oriented to direct an excision, wherein upstream andadjacent to R1′ is the DSC and wherein downstream of and adjacent to R1is R2. In a specific embodiment, the MFA comprises from 5′ to 3′, withrespect to the direction of transcription, R1, R2, R3, R4, the NSI inantisense orientation, R5, R2′ wherein R2/R2′ are oriented to direct aninversion, the DSC, R1′ wherein R1/R1′ are oriented to direct aninversion, the reporter gene, R3′ wherein R3/R3′ are oriented to directan inversion, the COIN in antisense orientation, R5′ wherein R5/R5′ areoriented to direct an excision, and R4′ wherein R4/R4′ are oriented todirect an excision.

In a specific embodiment, R1/R1′ are Rox sites, R2/R2′ are FRT or FRT3sites, R3/R3′ are FRT or FRT3 sites that are not the same as R2/R2′,R4/R4′ are lox2372 sites or loxP sites, and R5/R5′ are lox2372 sites orloxP sites that are not the same as R4/R4′.

In a specific embodiment, the MFA comprises a placement of recombinasesites and COIN, NSI, DSC, and reporter as shown in FIG. 12, Panel A.Treatment with a selected recombinase results in the allele shown inFIG. 12, Panel B. Alternate treatment with a different selectedrecombinase results in the allele shown in FIG. 12, Panel C. Treatmentof the allele of FIG. 12, Panel C with yet another different recombinaseresults in the allele shown in FIG. 12, Panel D.

In one aspect, a nucleic acid construct is provided, comprising a MFAcomprising, from 5′ to 3′ with respect to the direction oftranscription, a reporter in sense orientation, a DSC, an NSI inantisense orientation, and a COIN in antisense orientation; whereupontreatment of the MFA with a first selected recombinase, the reporter isexcised, the NSI is placed in sense orientation, and the COIN remains inantisense orientation, and wherein the allele comprises recombinasesites that allow for an inversion of sequence that upon treatment with asecond selected recombinase would place the COIN in sense orientationand the NSI in antisense orientation. In one embodiment, following withthe first selected recombinase, the allele is treated with the secondselected recombinase. In one embodiment, the COIN signals that the NSIhas been placed in antisense orientation following treatment with thesecond recombinase.

In one embodiment, the MFA comprises, from 5′ to 3′ with respect to thedirection of transcription, a recombinase site R1, a reporter, a secondrecombinase site R2, a DSC, a third recombinase site R3, an NSI inantisense orientation, a fourth recombinase site R4, a fifth recombinasesite R5, a sixth recombinase site R1′ that is compatible with R1 andthat is oriented with respect to R1 to direct an inversion, a seventhrecombinase site R3′ that is compatible with R3 and that is orientedwith respect to R3 to direct an inversion, a COIN in antisenseorientation, an eighth recombinase site R5′ that is compatible with R5and that is oriented with respect to R5 to direct an excision, a ninthrecombinase site R4′ that is compatible with R4 and that is orientedwith respect to R4 to direct an excision, and a tenth recombinase siteR2′ that is compatible with R2 and that is oriented with respect to R2to direct an excision. In a specific embodiment, R1/R1′ are FRT3 or FRTsites, R2/R2′ are Rox sites, R3/R3′ are FRT3 or FRT sites that aredifferent from R1/R1′, R4/R4′ are loxP or lox2372 sites, and R5/R5′ areloxP or lox2372 sites that are different from R4/R4′ sites.

In a specific embodiment, the MFA comprises a placement of recombinasesites and COIN, NSI, DSC, and reporter as shown in FIG. 13, Panel A.Treatment with a selected recombinase results in the allele shown inFIG. 13, Panel B. Treatment of the allele of FIG. 13, Panel B with adifferent recombinase results in the allele shown in FIG. 13, Panel C.

In one aspect, a nucleic acid construct is provided, comprising a MFAcomprising, from 5′ to 3′ with respect to the direction oftranscription, a COIN in antisense orientation, an NSI in antisenseorientation, a DSC, and a reporter in sense orientation; whereupontreatment of the MFA with a first selected recombinase, the reporter isexcised, the NSI is placed in sense orientation, and the COIN remains inantisense orientation, and wherein the allele comprises recombinasesites that allow for an inversion of sequence that upon treatment with asecond selected recombinase would place the COIN in sense orientationand the NSI in antisense orientation. In one embodiment, following withthe first selected recombinase, the allele is treated with the secondselected recombinase. In one embodiment, the COIN signals that the NSIhas been placed in antisense orientation following treatment with thesecond recombinase.

In one embodiment, the MFA comprises, from 5′ to 3′ with respect to thedirection of transcription, a recombinase site R1, a second recombinasesite R2, a third recombinase site R3, a COIN in antisense orientation, afourth recombinase site R4, a fifth recombinase site R5, a sixthrecombinase site R3′ that is compatible with R3 and that is orientedwith respect to R3 to direct an excision, a seventh recombinase site R2′that is compatible with R2 and that is oriented with respect to R2 todirect an excision, an NSI in antisense orientation, an eighthrecombinase site R4′ that is compatible with R4 and that is orientedwith respect to R4 to direct an inversion, a DSC, a ninth recombinasesite R1′ that is compatible with R1 and that is oriented with respect toR1 to direct an excision, a reporter in sense orientation, and a tenthrecombinase site R5′ that is compatible with R5 and that is orientedwith respect to R5 to direct an inversion. In a specific embodiment,R1/R1′ are Rox sites sites, R2/R2′ are loxP or lox2372 sites, R3/R3′ areloxP or lox2372 sites that are different from R2/R2′, R4/R4′ are FRT orFRT3 sites, and R5/R5′ are FRT or FRT3 sites that are different fromR4/R4′.

In a specific embodiment, the MFA comprises a placement of recombinasesites and COIN, NSI, DSC, and reporter as shown in FIG. 14, Panel A.Treatment with a selected recombinase results in the allele shown inFIG. 14, Panel B. Treatment of the allele of FIG. 14, Panel B with adifferent recombinase results in the allele shown in FIG. 14, Panel C.

In one aspect, a nucleic acid construct is provided, comprising a MFAcomprising, from 5′ to 3′ with respect to the direction oftranscription, an NSI in antisense orientation, a DSC, a reporter insense orientation, and a COIN in antisense orientation; whereupontreatment of the MFA with a first selected recombinase, the reporter isexcised, the DSC is excised, the NSI is placed in sense orientation, andthe COIN remains in antisense orientation, and wherein followingtreatment with the first selected recombinase the allele comprisesrecombinase sites that allow for an inversion of sequence that upontreatment with a second selected recombinase would place the COIN insense orientation and the NSI in antisense orientation. In oneembodiment, following with the first selected recombinase, the allele istreated with the second selected recombinase. In one embodiment, theCOIN signals that the NSI has been placed in antisense orientationfollowing treatment with the second recombinase.

In one embodiment, the MFA comprises, from 5′ to 3′ with respect to thedirection of transcription, a recombinase site R1, a second recombinasesite R2, a third recombinase site R3, an NSI in antisense orientation, afourth recombinase site R4, a fifth recombinase site R5, a sixthrecombinase site R2′ that is compatible with R2 and that is orientedwith respect to R2 to direct an inversion, a DSC, a seventh recombinasesite R1′ that is compatible with R1 and that is oriented with respect toR1 to direct an excision, a reporter in sense orientation, an eighthrecombinase site R3′ that is compatible with R3 and that is orientedwith respect to R3 to direct an inversion, a COIN in reverseorientation, a ninth recombinase site R5′ that is compatible with R5 andthat is oriented with respect to R5 to direct an excision, and a tenthrecombinase site R4′ that is compatible with R4 and that is orientedwith respect to R4 to direct an excision. In a specific embodiment,R1/R1′ are Rox sites, R2/R2′ are FRT or FRT3 sites, R3/R3′ are FRT orFRT3 sites that are different from R2/R2′, R4/R4′ are loxP or lox2372sites, and R5/R5′ are loxP or lox2372 sites that are different fromR4/R4′.

In a specific embodiment, the MFA comprises a placement of recombinasesites and COIN, NSI, DSC, and reporter as shown in FIG. 15, Panel A.Treatment with a selected recombinase results in the allele shown inFIG. 15, Panel B. Treatment of the allele of FIG. 15, Panel B with adifferent recombinase results in the allele shown in FIG. 15, Panel C.

In one aspect, a multifunctional allele is provided, comprising a DSC, areporter, a COIN, a NSI, and five pairs of recombinase sites arrangedamong the reporter, the DSC, the COIN, and the NSI, wherein no pair ofrecombinase sites is identical to any other pair, and wherein a firsttwo pairs of recombinase sites are recognized by the same firstrecombinase, a second two pairs of recombinase sites are recognized bythe same second recombinase, and the fifth pair of recombinase sites arerecognized by a third recombinase, wherein the first, second, and thirdrecombinases are not identical, and wherein, with respect to directionof transcription, the MFA comprises (from 5′ to 3′): (a) an actuatingsequence (e.g., with reporter) in sense orientation, the DSC in sense orantisense orientation, the NSI in antisense orientation, the COIN inantisense orientation; (b) the COIN in antisense orientation, the NSI inantisense orientation, the DSC in sense or antisense orientation, thereporter in sense orientation; (c) the NSI in antisense orientation, theDSC in sense or antisense orientation, the reporter in senseorientation, the COIN in antisense orientation; (d) the reporter insense orientation, the DSC in sense or antisense orientation, the NSI inantisense orientation, the COIN in antisense orientation; (e) the COINis in antisense orientation, the NSI in antisense orientation, the DSCin sense or antisense orientation, the reporter in sense orientation;or, (f) the NSI in antisense orientation, the DSC in sense or antisenseorientation, the reporter in sense orientation, the COIN in antisenseorientation.

In one embodiment, the arrangement is as in (a), and the pairs ofrecombinase sites are arranged such that upon exposure to the thirdrecombinase, the fifth pair of recombinase sites direct an excision ofthe DSC, the NSI, and the COIN, wherein the reporter is maintained insense orientation.

In one embodiment, the arrangement is as in (a), and the pairs ofrecombinase sites are arranged such that upon exposure to the firstrecombinase, a modified MFA forms wherein the first two pairs ofrecombinase sites direct excision of the reporter and excision of theDSC and inversion of the NSI to sense orientation, wherein the COIN ismaintained in antisense orientation. In a further embodiment, themodified MFA comprises the second two pairs of recombinase sites that,upon exposure to the second recombinase, result in an allele wherein theNSI is excised and the COIN is placed in sense orientation.

In one embodiment, the arrangement is as in (b), and the pairs ofrecombinase sites are arranged such that upon exposure to the thirdrecombinase, the fifth pair of recombinase sites direct an excision ofthe COIN, the NSI, and the DSC, wherein the reporter is maintained insense orientation.

In one embodiment, the arrangement is as in (b), and the pairs ofrecombinase sites are arranged such that upon exposure to the firstrecombinase, a modified MFA forms wherein the first two pairs ofrecombinase sites direct excision of the DSC and the reporter and directinversion of the NSI to the sense orientation, wherein the COIN ismaintained in antisense orientation. In a further embodiment, themodified MFA comprises the second two pairs of recombinase sites that,upon exposure to the second recombinase, result in an allele wherein theNSI is excised and the COIN is placed in sense orientation.

In one embodiment, the arrangement is as in (c), and the pairs ofrecombinase sites are arranged such that upon exposure to the fifthrecombinase, the NSI and the DSC are excised and the reporter and theCOIN are maintained in antisense orientation.

In one embodiment, the arrangement is as in (c), and the pairs orrecombinase sites are arranged such that upon exposure to the firstrecombinase, a modified MFA forms wherein the DSC and the reporter areexcised, and the NSI is placed in sense orientation, wherein the COIN ismaintained in antisense orientation. In a further embodiment, themodified MFA comprises the second two pairs of recombinase sites that,upon exposure to the second recombinase, result in an allele wherein theNSI is excised and the COIN is placed in sense orientation.

In one embodiment, the arrangement is as in (d), and the pairs ofrecombinase sites are arranged such that upon exposure to the fifthrecombinase, the DSC, the NSI, and the COIN are excised and the reporteris maintained in sense orientation.

In one embodiment, the arrangement is as in (d), and the pairs ofrecombinase sites are arranged such that upon exposure to the firstrecombinase, a modified MFA forms wherein the reporter and the DSC areexcised, and the NSI is placed in sense orientation, wherein the COIN ismaintained in antisense orientation. In a further embodiment, themodified MFA comprises the second two pairs of recombinase sites that,upon exposure to the second recombinase, result in an allele wherein theCOIN is placed in sense orientation and the NSI is placed in antisenseorientation.

In one embodiment, the arrangement is as in (e), and the pairs ofrecombinase sites are arranged such that upon exposure to the fifthrecombinase, the COIN, the NSI, and the DSC are excised and the reporteris maintained in sense orientation.

In one embodiment, the arrangement is as in (e), and the pairs ofrecombinase sites are arranged such that upon exposure to the firstrecombinase, a modified MFA is formed wherein the DSC and the reporterare excised, the NSI is placed in sense orientation, and the COIN ismaintained in antisense orientation. In a further embodiment, themodified MFA comprises the second two pairs of recombinase sitesarranged such that, upon exposure to the second recombinase, the NSI isplaced in antisense orientation and the COIN is placed in senseorientation.

In one embodiment, the arrangement is as in (f), and the pairs ofrecombinase sites are arranged such that upon exposure to the fifthrecombinase, the NSI and the DSC are excised, the reporter is maintainedin sense orientation, and the COIN is maintained in antisenseorientation.

In one embodiment, the arrangement is as in (f), and the pairs ofrecombinase sites are arranged such that upon exposure to the fifthrecombinase, the NSI and the DSC are excised and the reporter ismaintained in sense orientation and the COIN is maintained in antisenseorientation.

In one embodiment, the arrangement is as in (f), and the pairs ofrecombinase sites are arranged such that upon exposure to the firstrecombinase, a modified MFA is formed wherein the DSC and the reporterare excised and the NSI is placed in sense orientation and the COIN ismaintained in antisense orientation. In a further embodiment, themodified MFA comprises the second two pairs of recombinase sitesarranged such that, upon exposure to the second recombinase, the NSI isplaced in antisense orientation and the COIN is placed in senseorientation.

In one aspect, a method for making a cell that comprises a constructhaving a nucleotide sequence of interest in antisense orientation and aCOIN in antisense orientation is provided, comprising the step ofintroducing into a genome of a cell an MFA as described herein,identifying the cell comprising the MFA, followed by a step of exposingthe genome to a first recombinase, wherein action of the firstrecombinase on the construct in the genome results in the nucleotidesequence of interest being placed in the sense orientation.

In one embodiment, the cell is a pluripotent cell, an inducedpluripotent cell, a totipotent cell, or an ES cell. In a specificembodiment, the ES cell is a mouse or rat ES cell.

In one embodiment, the construct is introduced into the cell byhomologous recombination. In another embodiment, the construct israndomly integrated into a nucleic acid of the cell. In one embodiment,the nucleic acid of the cell is the cell's genome.

In one embodiment, the NSI comprises an exon. In one embodiment, the NSIcomprises an exon and flanking intronic sequence. In a specificembodiment, the flanking exon is flanked 5′ and 3′ with intronicsequence. In one embodiment, the nucleotide sequence comprises two ormore exons, and in a specific embodiment comprises intronic sequence(s).In another embodiment, the NSI lacks an exon, or lacks a fragment of anexon.

In one embodiment, the NSI is a wild-type exon or exons of a gene. Inanother embodiment, the NSI is an exon or exons of a gene having one ormore nucleic acid substitutions, deletions, or additions.

In one embodiment, the COIN comprises an exon of a gene that comprisesone or more nucleic acid substitutions, deletions, or additions. In aspecific embodiment, the COIN comprises an exon of a human, mouse,monkey, or rat gene.

In one embodiment, the COIN comprises a 3′ splice region. In a specificembodiment, the 3′ splice region is followed by a sequence selected froma cDNA, an exon-intron sequence, a microRNA, a microRNA cluster, a smallRNA, a codon-skipping element, an IRES, a polyadenylation sequence, anda combination thereof. In a specific embodiment, the small RNA is amirtron. In a specific embodiment, the codon-skipping element is T2A,E2A, or F2A.

In one embodiment, the COIN is selected from a reporter, a genetrap-like element (GT-like element), and a gene trap-like reporter(GT-like reporter). In a specific embodiment, the GT-like element isselected from SA-drug resistance cDNA-polyA. In a specific embodiment,the GT-like reporter is selected from SA-reporter-polyA.

In one aspect, a method is provided for placing a multifunctional allelein a mouse cell genome, comprising a step of introducing into a locus ina mouse cell a targeting construct comprising a first recombinable unitthat comprises (a) an actuating sequence (e.g., a nucleotide sequenceand/or a reporter); (b) a DSC; (c) a NSI in antisense orientation withrespect to the locus; (d) a COIN in antisense orientation with respectto the locus; and, (e) site-specific recombinase recognition sitesarranged in recombinable units for deleting the reporter and the DSC,for inverting the NSI back to the sense orientation, and for invertingthe COIN and deleting or re-inverting the NSI.

In one embodiment, the site-specific recombinase recognition sites arearranged in recombinable units such that the NSI will be re-invertedinto the antisense strand and the COIN will be inverted into the sensestrand. In another embodiment, the site-specific recombinase recognitionsites are arranged in recombinable units such that the NSI will bedeleted and the COIN will be inverted into the sense strand.

In one embodiment, the recombinable units are arranged such that uponexposure of the MFA-modified target locus to a first recombinase, afirst recombinable unit comprising the reporter and DSC are deleted andthe NSI is placed in the sense orientation with respect to the locus andthe COIN is maintained in the antisense orientation, forming a secondrecombinable unit.

In one embodiment, the nucleotide sequence of interest in antisenseorientation is an exon in the antisense orientation, or an exon flankedby intronic sequence wherein the exon and the intronic sequence are eachin antisense orientation. In a specific embodiment, the exon beingplaced in the antisense orientation is identical to the exon beingreplaced by the targeting construct. In a specific embodiment, the NSIin antisense orientation is an exon and sequence surrounding the exon.In a specific embodiment, the NSI is two or more exons. In a specificembodiment, the NSI is non-exonic sequence.

In one embodiment, the second recombinable unit generated by the actionof the first recombinase is exposed to a second recombinase, wherein thesecond recombinase deletes the NSI and places the COIN in the senseorientation.

In one embodiment, the second recombinable unit generated by the actionof the first recombinase is exposed to a second recombinase, wherein thesecond recombinase places the NSI in antisense orientation and placesthe COIN in sense orientation.

In one aspect, a method for complementation of a knockout is provided,comprising introducing into a nonhuman animal an MFA as describedherein, wherein the nucleic acid construct comprises a wild-type nucleicacid sequence in antisense orientation and a COIN in the antisenseorientation, wherein upon exposure of the nucleic acid construct to afirst recombinase the wild-type nucleic acid sequence inverts to senseorientation and is transcribed but the COIN remains in the antisenseorientation; and wherein upon exposure to a second recombinase the wildtype nucleic acid sequence is excised, or inverted back to the antisensestrand, and the COIN inverts to sense orientation.

In one embodiment, the nonhuman animal is a mouse.

In one embodiment, the COIN is a reporter element. In one embodiment thereporter element is selected from a fluorescent protein, a luminescentprotein, or an enzyme. In a specific embodiment, the reporter isselected from GFP, eGFP, CFP, YFP, eYFP, BFP, eBFP, DsRed, MmGFP,luciferase, LacZ, and Alkaline Phosphatase.

In one aspect, a mammalian cell comprising a multifunctional allele inaccordance with the invention is provided.

In one embodiment, the mammalian cell is selected from a mouse cell, anda rat cell. In one embodiment, the cell is selected from a stem cell, anembryonic stem (ES) cell, an induced pluripotent cell, a pluripotentcell, and a totipotent cell.

In one aspect, a non-human embryo or non-human animal comprising amultifunctional allele in accordance with the invention is provided.

In one embodiment, the non-human embryo or non-human animal comprises amultifunctional allele that has been exposed to one or moresite-specific recombinases. In a specific embodiment, themultifunctional allele has been exposed to the one or more site-specificrecombinases as the result of a breeding step wherein a non-human animalcomprising a multifunctional allele has been mated with a non-humananimal comprising the one or more site specific recombinases, and thenon-human embryo or non-human animal is a progeny of the breeding step.

In one aspect, a cell comprising an MFA as described herein is provided,wherein the cell is a mammalian cell, e.g., an ES cell or pluripotent orinduced pluripotent cell. In a specific embodiment, the cell is a mouseor rat cell.

In one aspect, a non-human animal is provided, comprising an MFA asdescribed herein, or an MFA that has been exposed to one or morerecombinases as described herein.

In one aspect, a non-human embryo is provided, comprising an MFA asdescribed herein, or an MFA that has been exposed to one or morerecombinases as described herein.

In one aspect, a cell, a non-human embryo, or a non-human animal madeusing an MFA as described herein is provided.

In one aspect, a cell, a non-human embryo, or a non-human animal madeusing an MFA as described herein is provided.

Any aspect or embodiment can be used in connection with any other aspector embodiment as appropriate, e.g., any reporter or DSC recited inconnection with any particular MFA embodiment can be used with any MFAembodiment described herein, and any particular recombinase orrecombinase site mentioned in connection with any particular MFAembodiment can be used with any MFA embodiment described herein.

Other embodiments are described and will become apparent to thoseskilled in the art from a review of the ensuing detailed description.

DETAILED DESCRIPTION

The invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. Theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

The phrase “sense orientation,” or “sense,” refers to the codingdirection or sense strand of a transcribable nucleic acid sequence inthe local context of the genome, e.g., when a sequence is placed in“sense orientation” in or near a transcribable sequence in a genome, theorientation of the sequence is compatible with transcription and, forprotein-coding genes also translation of the sequence in the region orlocus or gene in which the sequence is placed. The phrase “antisenseorientation,” or “antisense,” refers to placement of a sequence at aregion or locus or gene in which the sequence is in the strand opposite(or antisense) to that which is compatible with transcription. Thus, ina specific example, if a sequence is placed in “sense” orientation in agene, it can generally be transcribed. If a sequence is placed in“antisense” orientation, it generally will not be transcribed. For lociwhere transfer between sense and antisense strands might result intranscription from either strand, the sequence can be selected orengineered such that transfer from sense to antisense or from antisenseto sense would result in transcription from one strand, but not either.

The term “COIN” includes reference to a conditional element. Aconditional element comprises a nucleotide sequence whose expression (orfailure to express) is contingent upon the occurrence of an independentevent. For example, a coding region that in a sense orientation wouldeither encode a protein or fragment thereof or a non-coding RNA (ncRNA)is placed in antisense orientation flanked on both sides bysite-specific recombination sites in opposite orientation. In theabsence of a site-specific recombinase that recognizes the flankingsites, the coding region is not transcribed, because it is placed in theantisense strand with respect to the target gene. Upon treatment withthe cognate site-specific recombinase, the COIN sequence is inverted,and as a result it becomes incorporated into the transcribed message,resulting in expression of the protein or fragment thereof or in the ofthe ncRNA.

The term “incompatible,” when used to describe two or more recombinaserecognition sites, refers to the quality that the two or morerecombinase recognition sites cannot be recombined with one another (butthe two or more recombinase recognition sites can be recombined withother cognate (e.g., identical) recombinase recognition sites).

Knockouts and Conditional Alleles

The study of gene function by genetic methods has relied on thediscovery of naturally occurring variants or mutant alleles, or on thedeliberate generation of such variants and mutant alleles. The latterhas proceeded either by the random mutagenesis followed byphenotype-based screens and then elucidation of the causative mutation—aprocess that has been referred to as “forward genetics”, or by geneticengineering methodology whereby mutations are rendered in specific“target” genes or loci—an approach that has been referred to as “reversegenetics.”

In the mouse—the most widely used mammalian model organism—the abilityto engineer specific, molecularly extremely well defined mutations viagene targeting has dominated the field of reverse genetics. However, themajority of variants made to date have been relatively simple nullalleles, commonly referred to as “knockout alleles” or simply“knockouts”, and usually encompass a deletion of the exon-intron regionof a gene or part thereof, and in more recent years with concomitantreplacement of that region with a reporter cDNA, such as LacZ. Theadaptation of site-specific recombinases and their cognate recognitionsites (such as Cre/Iox, Dre/Rox, PhiC31\int/attP-attB, Flp/FR7), derivedfrom bacteriophages or yeast and modified for use in mammalian cells, isa more recent development that has not only made possible thepost-targeting excision of the DSC (as long as it is flanked bysite-specific recombinase recognition sites) but has also enabled theengineering of conditional-null alleles. Conditional-null alleles havebeen developed wherein the exon-intron region of the target gene—or morefrequently a part thereof—is flanked by recombinase recognition sites,rendering the modified allele amenable to conversion to the null stateby the action of the cognate recombinase. The advantage of this methodover regular knockouts is that the conversion of the modified gene to aknockout can be spatio-temporally controlled by controlling the place(organ, tissue, or cell type), time, and sometimes, also the durationthat the cognate recombinase will be active.

Traditionally, conditional-null alleles have been engineered as afollow-up to the corresponding simple knockout alleles, mostly in caseswhere the latter is either embryonic lethal and/or displays a pluralityof phenotypes, hence rendering the study of the target gene's functionimpossible in an adult setting (in the case of embryonic lethality), orhard to interpret in a specific cell type or biological process (in thecase where the gene displays a plurality of phenotypes). Given theamount of effort, time, and expense that it takes to generategenetically modified mice via gene-targeting, this step-wise fashion offirst generating a knockout, then deciphering its phenotype, and thenengineering a conditional-null, has been considered burdensome by anincreasing number of investigators. In addition, for a small number ofgenes, regular knockout alleles cannot go through the germline as theyresult in embryonic lethality even at the heterozygous null state.Therefore, the desire to be able to engineer dual (null and conditional)or even multi-modality alleles in a single gene-targeting step has beena persistent goal of those involved in the art as well as the communityof end users. Two methods have indeed tried to address this need: FlExand Knockout-first (KO-first).

The FlEx method has been used both for targeting and as a gene trap(GT), but the basic design principles are the same irrespective of thefinal application. A basic design of a FlEx is shown in FIG. 1, with Urepresenting, e.g., a DSC and D representing a reporter. The result ofrecombinase action on the FlEx construct is permanent deletion of the Uelement (e.g, the DSC) and inversion (and expression) of the D element(e.g., the reporter).

In its original embodiment and application, FlEx was developed as amethod to engineer conditional alleles. FlEx was first used to generatea “conditional-null” allele for Rarg, by inserting the FlEx cassetteinto this gene such that a loxP/lox511 couplet was inserted upstream ofexon 8 of Rarg, and the remainder of the FlEx cassette—composed 5′ to 3′of SA-lacZ-SV40polyA (a GT-like element) in the antisense orientationwith respect to Rarg, and then another loxP/lox511 couplet in theantisense orientation with respect to the first loxP/lox511 couplet, andcontaining a neomycin phosphotransferase mini gene (neo) in the senseorientation—into intron 8 of Rarg. This design empowers Cre-mediatedinversion of the GT-like element SA-lacZ-SV40polyA such that it isbrought into the sense strand and acts as a gene trap; simultaneously,exon 8 of Rarg is brought into the antisense orientation (effectivelyensuring that even in the case where transcription does not terminate atthe end of the GT-like element SA-lacZ-SV40polyA, exon 8 will not beincorporated into the read-through message), while neo is simultaneouslydeleted, and thereby resulting in a null allele of Rarg in which theexpression of Rarg is replaced by that of lacZ.

However, in spite of the success of this method in generating a nullallele (by exposure to Cre), the unrearranged (pre-Cre) FlEx allele ofRarg was not a true conditional-null, as originally designed, but rathera severe hypomorphic allele, where the expression of Rarg^(FLEx) wassignificantly reduced in comparison to Rarg. As a result,Rarg^(FLEx/FLEx) mice displayed a phenotype that resembled a less severeform of the Rarg knockout mice, and revealing the inability of thisinitial embodiment of FlEx to generate a true conditional-null allele.

The FlEx method has been also adapted for use in gene trapping. In thatvariation of the method, a GT element (SA-βgeo-polyA, where βgeo is anin-frame fusion of lacZ with neo open reading frames, hence combiningthe ability to report via LacZ and select via Neo) was flanked by twoFlEx-like arrays, an outer array composed of an FRT/FRT3 couplet, and aninner array composed of an loxP/lox511 couplet, both in mirror imageconfiguration with respect to one another. In this manner, successfulincorporation of the resulting GT vector into actively transcribed geneswould result in expression of βgeo and hence allow selection for theseevents by selecting for G418, and depending on the site of incorporationof the GT element theoretically also result in the generation offunctional null alleles for the corresponding genes. Once a gene hasbeen trapped to generate the corresponding FlEx allele, the resultingallele may be a knockout allele or a hypomorphic allele, i.e., one wherethe expression of the trapped gene is downregulated. Treatment of theseFlEx alleles with Flp recombinase should in principle invert the GTelement to the anti-sense strand, thereby alleviating transcriptionaltermination within the trap element, and hence converting the modifiedgene to conditional GT. This conditional GT, now “hidden” in theantisense strand, can be reactivated by exposure to Cre, which willre-invert it by acting on the loxP/lox511 couplets of the FlEx array.

This application of FlEx technology relies on a GT element to generatenull alleles. It is therefore subject to the limitations of genetrapping technology, which does not guarantee that a true knockout willbe generated and takes on the additional risk of inactivating regulatoryelements (by random insertional inactivation). Both the placement of theGT element as well as the degree that it is effective in terminatingtranscription can impact whether any given allele will be a null allele.An additional problem is that for the majority of genes that do not havean already established function, and more so one that links the gene toa phenotype determined through the study of a definitive null allele, itis very difficult to prove conclusively that a GT allele is truly a nullallele. In fact, for these as well as other, mostly technical reasons,after 4 years of adoption and use by a large scale mouse mutagenesisconsortium—EUCOMM—the FIEX-based gene trapping method has been abandonedin favor of gene targeting using the KO-first method.

Similar to the FlEx method, typical current KO-first alleles rely atleast in part on a GT-like element (either SA-LacZ-polyA orSA-βgeo-polyA) to generate a knockout-like allele. However, inrecognition of the limitations that have been associated with thatapproach (effectively learning from the experience gained with GTs, aswell as theoretical considerations), KO-first also requires that thefloxed critical exon downstream of the GT-like element must be deleted(using Cre) in order to generate a true null allele. Therefore, inpractice, this method first requires placement of a FRT-flankedreporter/GT-like cassette plus a drug mini-gene into an intron of thetarget gene somewhere upstream of the exon to be deleted, whilesimultaneously floxing the exon slated to be deleted. This exon has beenreferred to as the “critical exon”, and irrespective of the criteriathat are used to define “critical exon”, the KO-first method clearlyrequires its removal in order to render the resulting allele a truenull. Therefore, following targeting, the resulting allele is neither atrue null nor a conditional-null allele. The reasons that the resultingallele is not a true null has been attributed to the fact that withoutremoval of the critical exon (which is floxed) by Cre, there remains thepossibility of read-through transcription and splicing around of theGT-like cassette, as well as transcription of the gene's messagedownstream of the GT-like cassette due to the presence of the drugmini-gene. The reason that the resulting allele is not aconditional-null allele lies in the fact that without removal of boththe reporter/GT-like cassette and the drug mini-gene, generation of thenormal message (normal composition, as well as level and sites ofexpression) cannot take place.

Therefore, depending on the desired use—null orconditional-null—KO-first alleles must be subjected to a secondpost-targeting step. In the case where a true null is desired, theKO-first allele must be treated with Cre recombinase to delete thecritical exon (which is floxed).

Conversely, a conditional allele can be generated after a Flp-mediatedremoval of the reporter/GT-like cassette and the drug mini-gene, whichare together flanked by FRT sites. In this manner, the onlymodifications that remain are an FRT site and the floxed “critical”exon. This allele in turn can be converted to null by Cre-mediatedremoval of the floxed exon.

Although the KO-first method addresses some of the limitations of FlEx,it is still hampered by three main drawbacks that limit its utility:first, although it rectifies the lack of reliability of GT-like elementsto generate a true KO-first, it fails to provide a true KO-first withoutan additional post-targeting step; second, due to the criteria used todefine “critical exons”, KO-first is limited to protein-coding genes,effectively placing out of reach all the non-protein coding genes (i.e.,those that encode ‘non-coding’ RNAs, a class of the very importantbiomolecules). Furthermore, of the protein-coding genes only those forwhich a “critical exon” can be defined are amenable to the KO-firstdesign. The criteria for defining a critical exon are that its deletionresults in a frame sift between the part of the open reading frame (ORF)preceding it and the part of the ORF following it. This is becauseinduction of this frame shift is obligatory for the KO-first method toprovide a definitive knockout. Therefore, even certain classes ofprotein-coding genes are not amenable to KO-first design. These includegenes where the ORF is contained within one exon, and genes where all orthe majority of tandem exons leave off in the same frame. Thirdly, oncethe KO-first allele has been converted into a conditional-null allele(by the action of Flp), the resulting allele does not provide anymechanism for affirmative reporting of nuliness upon conversion of theconditional allele to the null state. Typically, knockout-first allelesremove the reporter (e.g., lacZ) together with the DSC using (a stepaccomplished using Flp recombinased, as the SA-lacZ-polyA and DSC usedin KO-first are FRTed) leaving behind only the floxed exon (plus a FRTsite upstream of it). Thus, no option exists in conventionalknockout-first approaches for a reporter function to report achievementof a null allele.

Multifunctional Alleles

A multifunctional allele (MFA) approach is provided that permits removalor inactivation of a nucleotide sequence in a genome by introduction ofa set of functional elements that comprise an actuating sequence (whichconfirms removal or inactivation), resulting in a true knockout, andthat also contains one or more conditional elements whose expression inthe allele is conditional (i.e., dependent on certain molecular eventsor cues) and reportable.

The MFA approach provides targeting options to generate trueknockout-first alleles that do not require a second post-targeting stepto convert the targeted alleles to null status, thus providing anadvantage and conceptual breakthrough over typical currentknockout-first alleles that require a post-targeting step to converttargeted alleles to null alleles. The MFA approach is also not limitedto use with “critical exons” and not limited to knockouts byframeshifts, but is generally applicable for modifying any nucleotidesequence of interest. MFAs provide enhanced versatility and amultiplicity of allele options following a single targeting step.

The MFA approach provides a true KO-first allele that provides theopportunity for creating a second state in the recipient genome uponinversion of any selected sequence linked with an actuating sequence,and wherein upon inversion transcription of the selected sequence can bereported. The selected sequence can be, e.g., a COIN, which, withoutlimitation, can itself comprise an actuating sequence that, e.g.,comprises a repressor to control transcription of another gene orregulatory sequence. In one example, a single targeting step placing anMFA at a locus can allow modification of a wild-type locus to aparticular state, State A (e.g., a knockout of an endogenous gene). TheMFA is designed to enable a change of state to another particular state,State B (e.g., reinstatement of a wild-type phenotype), through theaction of a first recombinase. State B can be converted to State C(e.g., reestablishment of the knockout and expression of a COIN) by asecond recombinase, and so on. Thus, varying states at a selected locuscan be achieved from a single initial allele when employing MFAs.

The MFA approach can be used to place an MFA as a gene trap, such thatthe transgene comprising the MFA obtains expression of MFA elementsemploying an endogenous transcriptionally active promoter.

The MFA approach can be used in traditional transgenesis applicationswherein the actuating sequence comprises a promoter, exon, or exons andintrons, and optionally transcriptional control elements and followed bya DSC (optional, as it is not necessary for traditional,pronuclear-injection based transgenesis), an NSI (that can be a secondactuating sequence) in the antisense orientation with respect to thefirst actuating sequence, and a COIN (that can be a third actuatingsequence) also placed in the antisense orientation with respect to thefirst actuating sequence.

The MFA approach provides a multiplicity of options for creating locithat contain null alleles, conditional null alleles, COINs, actuatingsequences (which can include reporters) and DSCs, and other elements, ina single targeting step. Post-targeting manipulations of the locusprovide options through the use of recombinable units introduced intothe MFA-containing locus with the MFA construct in the single targetingstep. The number of different recombinases required to exercise thevarious manipulable options at the locus post-targeting is reduced byemploying different pairs of cognate site-specific recombinaserecognition sites that are incompatible (e.g., a pair of FRT sites and apair of FRT3 sites, a pair of loxP sites and a pair of lox2372 sites,etc.). Thus, exposure of an MFA-containing locus to a single recombinasecan independently act on at least two different recombinable units, torecombine a unit such that the unit places elements of interest (e.g.,reporters, DSCs, exons, COINs, etc.) in desired orientations, as well asre-orienting site-specific recombinase recognition sites within therecombinable unit to form new recombinable units.

The MFA approach provides an option for a COIN that can contain anydesired sequence, including but not limited to a reporter, or a cDNAencoding a mutant or variant form of the target gene or part of thetarget gene, or relatives and homologs of the target gene, or evennon-protein coding sequences such as microRNAs or clusters of microRNAs,or any combinations of these elements (as they can be accommodated bythe placement of internal ribosome entry sites or “self-cleaving”peptides—depending on the choice of elements—between the differentelements).

The MFA approach provides an option wherein knockout is achieved bytargeting, a first recombinase is employed to reestablish the knockedout element back into an active (wild type) state, and a secondrecombinase is employed to reestablish the knockout, wherein a COIN isplaced in sense orientation concomitant with reestablishment or knock-inof the knocked out element, thus reporting the reestablishment of theknockout in the cells where the second recombinase has been activated.

Although current knockout-first approaches lack a mechanism forreporting nullness upon conversion of a conditional allele to null, theMFA approach provides an option for a reporting element (e.g., a COIN,see FIG. 5 and FIG. 6, at bottom; detectable by genotyping and/or byvisualization or other qualitative or quantitative determination,optionally at the cell level) that, upon action of a second recombinaseinverts or inverts and excises a nucleotide sequence of interest (e.g.,an exon and surrounding sequence, NSI in FIG. 5 and FIG. 6) and placesthe reporting element in sense orientation, effectively reporting theinversion and/or excision of the NSI. In this embodiment, a null allelefollowing a single targeting step is converted, by a first recombinaseto a restored allele (in this embodiment, NSI of FIG. 5 and FIG. 6 is anexon and surrounding sequence or gene or part thereof replaced by thetargeting vector), and by a second recombinase to a null allele thatreports its presence by placement of the COIN in sense orientation.

Thus the MFA approach provides an option for assessing a phenotypiceffect of a knockout (following the targeting step), then exposing to afirst recombinase to re-establish the knocked out exon or gene or regionthereof, assessing the phenotypic effect of the reestablishment—i.e.,conversion back to wild type (a step equivalent to a complementationassay but devoid of the requirement of generating a new transgenic mouseline, a requirement which has traditionally accompanied complementationanalysis), then optionally exposing to the second recombinase toreestablish the knockout and assessing the phenotypic effect ofreestablishing the null allele. Thus, the MFA allele combines a trueknockout-first approach with the versatility of additional (conditional)elements, and the ability to conduct a true complementation-typeanalysis in a genetically modified animal in a protocol that comprises asingle targeting step.

In one MFA application, a method is provided for a complementationassay, comprising targeting an endogenous allele of a cell with an MFAin accordance with the invention, then in a post-targeting step,generating a conditional-null allele from the MFA (by exposure to afirst recombinase), wherein the nucleotide sequence of interest in theMFA comprises an exon or an exon plus surrounding sequence, or anotherregion of interest (associated with, e.g., a phenotype) in the senseorientation, and assessing a phenotypic effect of the conditional-nullallele (which should be wild type). In a further embodiment, the MFA isfurther exposed to a second recombinase that reestablishes nuliness, andoptionally a phenoypic effect is again measured. In a specificembodiment, the second recombinase also places a conditional reporter(e.g., a COIN) in the sense orientation, wherein the conditionalreporter reports conversion of the conditional-null allele to a nullallele or, as the case may be, reports the reestablishement of nuliness.

In one embodiment, the NSI comprises an exon and neighboring intronicsequence, or an exon-intron region of a target gene. In anotherembodiment, the NSI comprises a region encoding an ncRNA, microRNA,microRNA cluster, or other small ncRNA(s).

MFAs are alleles that can be placed randomly or targeted at a locus ofchoice in a genome. The MFA is engineered to produce null, conditional,or combination conditional/null alleles by a judicious placement of thesequences among an array of pairs of cognate site-specific recombinaserecognition sites. Resulting alleles produced by the placement ofconstructs in a genome are manipulable by selected recombinases, whichcan be introduced to the construct in the genome transiently or throughbreeding of an animal comprising the construct in its genome with ananimal comprising a gene for a selected recombinase (e.g., a Cre-, Fip-,or PhiC31\int-expressing strain).

In various embodiments, methods and compositions are provided forgenerating a true knockout-first allele where nullness does not dependupon carrying out a second step such as, e.g., removing a “criticalexon,” or “critical region,” by the action of a recombinase.Accordingly, embodiments are provided for generating in a singletargeting step an allele that is multifunctional, in that it is a trueknockout-first allele with a reporter, achieved in a single targetingrecombination step.

The methods and compositions for generating knockout alleles by the MFAapproach are not limited by a requirement to generate a frameshift viadeletion of inversion of a critical exon to generate a null allele, asrequired by some types of knockout alleles (e.g., KO-first or someembodiments of FlEx). Instead, the MFA method relies on its ability toremove the NSI from the transcriptional unit of the target gene at thetime of targeting, while simultaneously replacing the expression of theNSI with that of an actuating sequence. The actuating sequence cancomprise a GT-like element (e.g., a reporter such as SA-lacZ-polyA), acDNA, an exon or exons, regulatory elements (e.g., enhancers,insulators, operators). Since the actuating sequence isexperimenter-defined, alleles other than null can equally well berendered. For example, the actuating sequence may encode for adominant-negative or a constitutively active or an activated form of agene.

In various embodiments the MFA comprises a nucleotide sequence ofinterest and a COIN that are each in antisense orientation in theresulting allele, and further comprising an actuating sequence and/or aDSC both in sense orientation in the allele (or in sense and antisenseorientations, or each independently in sense or antisense orientation),whereupon following exposure to a first recombinase the actuatingsequence and/or DSC are deleted, the nucleotide sequence of interest isinverted to a sense orientation, and the COIN is maintained in theantisense orientation. The allele further comprises recombination sitespositioned so as to allow for subsequent simultaneous inversion by asecond recombinase of the nucleotide sequence of interest and the COIN,such that upon action of the second recombinase the nucleotide sequenceof interest is placed in antisense orientation and the COIN is placed insense orientation. In a further embodiment, the nucleotide of interestis deleted upon treatment with the second recombinase, leaving the COINin sense orientation. In a specific embodiment, the COIN is a reporteror a DSC. In another specific embodiment the nucleotide of interest isan exon or region of interest of a gene of one specie (e.g., mouse, rat,non-human primate, or human exon) and the COIN is an exon of a gene ofanother specie (e.g., a mouse, rat, non-human primate, or human exon).

The MFA approach also allows for a gene trap approach. In thisembodiment of the MFA approach, an MFA is inserted at atranscriptionally active locus. This may be achieved by randomrecombination, or by “targeted trapping” (see, e.g., U.S. Pat. No.7,473,557, hereby incorporated by reference). An actuating sequencepreceded by a splice acceptor and splice region and followed by a polyAsignal affords a knockout or “knockdown” of any existing transcribedgenomic sequence. Inclusion of a promoterless DSC, i.e. one whoseexpression is dependent on insertion within the sense strand of atranscriptionally active locus, assures positive selection of cellscontaining the MFA. Inclusion of a nucleotide sequence of interest (NSI)in antisense orientation, along with a COIN in antisense orientation, inconjunction with a recommended arrangement of site-specific recombinaserecognition sites, affords the ability to conditionally express the NSI(from the promoter of the trapped locus), upon exposure to a firstrecombinase. Then, upon exposure to a second recombinase, the expressionof the NSI can be turned off and simultaneously replaced by that of theCOIN. The promoterless DSC will ensure that any cell selected will havethe ability to express the promoterless NSI and the promoterless COIN,and that expression will be in accordance with the endogenous pattern ofexpression from the transcriptionally active locus.

Certain advantageous approaches using MFAs are conveniently described inconnection with particular embodiments (i.e., with reference to allelescomprising specific named recombinase sites and nucleotide sequences asshown in the figures) for convenience and not by way of limitation,i.e., suitable recombinases and recombinase recognition sites, actuatingsequences, reporters, DSCs, and nucleotide sequences of interest can beroutinely chosen based upon the disclosure herein. The “nucleotidesequence of interest” or “NSI” can be any nucleotide sequence ofinterest, e.g., an exon, an exon plus flanking sequence(s), two or moreexons, a fragment of a coding sequence, an entire coding sequence, aregulatory element or sequence, an non-protein coding sequence, anintron, or any combinations thereof, etc. COINs can comprise cDNAs aswell as non-protein coding sequences and may incorporate elements suchas polyadenylation signals and sites, microRNAs or other non-proteincoding RNAs, IRESs, codon-skipping peptides, and any combinationthereof. Certain COINs and some systems for using them can be found,e.g., in U.S. Pat. No. 7,205,148.

Methods and compositions for making and using MFAs in any cell,including non-human animal cells, and in non-human animals, areprovided. The methods and compositions can be employed using homologousrecombination (or random integration) to place useful alleles at anyselected site (or random site) in the genome of a cell. The methods andcompositions can be used in pluripotent, induced pluripotent, andtotipotent cells. Suitable cells for use with the methods andcompositions include ES cells, e.g., mouse or rat ES cells. In variousembodiments, true KO-first alleles are provided that afford an optionfor a conditional functionality with an embedded reporter function.

An example of how an arrangement of elements and recombinase recognitionsites can be designed to create a construct that will ablate thefunction of the target gene (i.e., create a null allele), or alter thefunction of the target gene (e.g. turning it into a dominant-negative,constitutively active, or hypomorphic allele), while at the same timeembed all the downstream elements that will allow (a) the generation ofa conditional allele, and (b) it reversion to a null with a reporter, isillustrated in FIG. 2.

FIG. 2 shows an embodiment of an MFA that can be placed into a genome(e.g., using homology arms to the left and right of the MFA shown).Post-targeting, the resulting allele can be converted to a conditionalallele, which is accomplished by deleting a first selected sequence andinverting a second selected sequence. The deletion and inversion can beachieved by the same recombinase or a different recombinase. Forexample, two pairs of incompatible Flp recognition sites can be used—oneto direct deletion and the other to direct inversion. One example of twosuch Flp sites are FRT sites and FRT3 sites. In another example, twopairs of incompatible Cre sites can be used, e.g., loxP and lox2372—oneto direct deletion and the other to direct inversion. Further, twodifferent recombinases can be used (e.g., a pair of loxP sites with Creand a pair of FRT sites with Flp). Any suitable sites can be chosen forthis embodiment, so long as the sites can direct deletion and inversionof recombinase site pairs of the MFA shown in FIG. 2 (specificembodiments of which are shown in FIG. 5 and FIG. 6).

Although the construct design of FIG. 2 can be used with any sequencesof interest (i.e., NSI is any sequence of interest), the constructdesign can be particularly useful to replace an exon of interest with amodified exon.

In one embodiment, NSI is a naturally occurring exon (or exons), and theCOIN is a modified exon (e.g., an exon comprising a mutation). The MFAis placed into a genome of, e.g., a mouse ES cell by, e.g., homologousrecombination (using appropriate mouse homology arms), and the ES cellis employed to make a genetically modified mouse that comprises theconstruct in the mouse germline. In one embodiment, change of state fromthe naturally occurring exon to the modified exon is achieved by theaction of a recombinase on the MFA.

In one embodiment, the construct is placed in a genome of, e.g., amouse, and the mouse either further comprises a recombinase (e.g., Cre)whose activity can be regulated. A recombinase can be regulated by,e.g., employing a fusion protein placing the recombinase under controlof an effector or metabolite (e.g., CreER^(T2), whose activity ispositively controlled by tamoxifen), placing the recombinase undercontrol of a tissue-specific promoter, or placing the recombinase undercontrol of a promoter (or other regulatory element) that is active at aparticular developmental stage (e.g., a Nanog promoter), or an induciblepromoter (e.g., one whose activity is controlled by doxycycline and TetRor TetR variants), or combinations of these technologies.

The MFA embodiment shown in FIG. 2 bears elements comprising a sequenceencoding an actuating sequence, a DSC, a nucleotide sequence of interest(NSI), and a COIN, wherein the elements are arranged among an array ofrecombinase recognition sites that are selected so as to provide adesired functionality to the MFA.

The top of FIG. 2 illustrates a nucleotide sequence of interest (NSI) ina genome of choice (e.g., an NSI in a mouse genome). An MFA as shown isintroduced into the genome by, e.g., homologous recombination to replacethe NSI. The NSI is replaced with the MFA shown, where the NSI of theMFA is inverted as shown and thus no longer incorporated into thetranscript of the target gene. The presence of the MFA can beconveniently confirmed if the actuating sequence contains a reporter(e.g., a lacZ). A DSC is present as well, to assist in selectingmodified cells (e.g., mouse ES cells modified with the MFA).

The MFA embodiment of FIG. 2 comprises five distinct units of sequence,defined by five sets of recombinase recognition sites. FIG. 3 contains aconceptual rendering of the five distinct units of sequence flanked bycompatible recombinase recognition sites.

The first distinct recombinable unit comprises R1/R1′ sites (e.g., FRT3sites) in opposite orientation (i.e., directing an inversion), whereinbetween the R1/R1′ sites the following are arranged: an actuatingsequence (a 3′ splice region and acceptor 5′ with respect to theactuating sequence, and a polyA signal 3′ with respect to the actuatingsequence, are not shown in FIG. 3 for the sake of simplicity), an R2site (e.g., a Rox site), a DSC, an R3 site (e.g., a FRT site) in thesame orientation as the R1 site, an R4 site (e.g., a loxP site), and anucleotide sequence of interest (NSI) in antisense orientation withrespect to direction of transcription (i.e., encoded by the antisensestrand) of the target gene, and an R5 site (e.g., a lox2372 site) in thesame orientation as the R4 site. Where an R3′ site (in oppositeorientation of the R3 site shown in FIG. 3A) is further includeddownstream of the 3′ R1′ site, the unit in the presence of a recombinasethat recongizes R1/R1′ will invert the NSI into a position fortranscription and delete the actuating sequence and DSC. In oneembodiment, a further sequence includes a COIN placed on the antisensestrand and followed by a R4′ site (e.g., loxP site) that is in oppositeorientation with respect to the R4 site of the unit, such that uponexposure to a recombinase that recognizes R4/R4′ (e.g., Cre), the COINis inverted such that the coding sequence of the COIN is now in positionfor transcription downstream of the NSI.

The second distinct recombinable unit (FIG. 3B) comprises R2/R2′ sites(e.g., Rox sites) in the same orientation (i.e., directing a deletion),comprising the following sequences disposed between the R2/R2′ sites: aDSC, a first R3 site (e.g., a first FRT site) in the same orientation asthe R1 site of the first distinct recombinable unit, a first R4 site(e.g., a first loxP site), an NSI in inverted (i.e., antisense)orientation with respect to the target gene, a first R5 site (e.g., afirst lox2372 site) in the same orientation with respect to the R4 site,an R1′ site (e.g., a second FRT3 site) and an R3′ site (e.g., a secondFRT site) both in opposite orientation as the R3 site, a COIN (inantisense orientation with respect to transcription of the target gene),an R5′ site (e.g., a second lox2372 site) in the same orientation as theR5 site, and an R4′ site (e.g., a second loxP site) in the sameorientation as R4. This second distinct recombinable unit is excisableby a recombinase that recognizes R2/R2′. When included in the MFA, thisunit can be excised to leave behind an actuating sequence (e.g., in someembodiments a reporter, e.g., a sequence encoding lacZ), flanked by anR1 and an R2 or R2′ site.

The third distinct recombinable unit (FIG. 3C) comprises R3/R3′ sites(e.g., FRT sites) in opposite orientation (i.e., directing aninversion), comprising the following sequences disposed between theR3/R3′ sites: an R4 site (e.g., a loxP site), a NSI in inverted (i.e.,antisense) orientation with respect to transcription of the target gene,an R5 site (e.g., lox2372 site) in the same orientation as the R4 siteof the unit (i.e., of FIG. 3C), and an R1′ site (e.g., a FRT site) inthe opposite orientation as the R3 site. This unit can be inverted bythe action of a recombinase that recognizes R3/R3′ (e.g., a Flprecombinase where R3/R3′ sites are FRT sites), resulting in placement ofthe NSI in proper orientation for transcription and translation.

The fourth distinct recombinable unit (FIG. 3D) comprises R4/R4′ sites(e.g., two loxP sites) in the same orientation (i.e., directing adeletion), comprising the following sequences disposed between theR4/R4′ sites: an NSI in inverted (i.e., antisense) orientation, an R5site (e.g., a first lox2372 site) and an R1′ site (e.g., a FRT3 site)and an R3′ site (e.g., a FRT site) each in the same orientation withrespect to the R4 site, a COIN (in antisense orientation), and an R5′site (e.g., a second lox2372 site) in the same orientation as the R5site. In the presence of a recombinase that recognizes R4/R4′ (e.g., Creif R4/R4′ are loxP sites, e.g.), this unit is excisable. If placedwithin the MFA and exposed to the R4/R4′ recombinase (in the absence ofexposure to a recombinase that recognizes R1/R1′, R3/R3′), this unitwill be deleted and leave behind the actuating sequence (e.g., in someembodiments a reporter, e.g., a sequence encoding lacZ) and the DSC.Thus, this unit allows for an embodiment in which the MFA, whenreplacing a sequence in a genome (e.g., replacing an exon), can act inthe presence of a recombinase that recognizes R4/R4′ as a null allelecomprising an actuating sequence and a DSC. The DSC of the MFA can beremoved, if desired, upon the action of a recombinase that recognizesR2/R2′ (e.g., a Dre recombinase where R2/R2′ are Rox sites) because theDSC would be flanked upstream and downstream with R2/R2′ sites in thesame orientation.

The fifth distinct recombinable unit (FIG. 3E) comprises R5/R5′ sites(e.g., two lox2372 sites) in the same orientation (i.e., directing adeletion) as well as in the same orientation of the R4/R4′ sites of thefourth distinct recombinable unit, comprising the following sequencesdisposed between the R5 and R5′ sites: an R1′ site (e.g., a FRT3 site)and an R3′ site (e.g., a FRT site) in the same orientation with respectto each other but in opposite orientation to the R1 site of the firstdistinct recombinable unit, and a COIN (in antisense orientation) withrespect to transcription of the target gene.

As those of skill in the art would recognize, the overlappingrecombinable units are so described to convey the structure of the MFA,rather than to limit the possible recombinable elements in the MFA. Forexample, those skilled in the art will recognize that each recombinableunit comprises site-specific recombination sites within the recombinableunit, and that action of a recombinase on sites (across recombinableunits) achieves desired and described manipulations of the MFA thatachieve intended functions of the MFA. For example, with reference toFIG. 3, the action of a recombinase that recognizes R1/R1′ and R3/R3′functions to manipulate portions of all five recombinable units as theyare conceptually displayed in FIG. 3.

Once an MFA is placed at a desired location in a genome it can beengineered such that it provides a null allele with a reportingfunction, wherein the null allele can be remodified (in post-targeting,recombinase-mediated step) such that it lacks all sequences flanked withrecombinase recognition sites oriented in the same direction. An exampleof this embodiment is shown in FIG. 4 showing examples of suitablerecombinase recognition sites, where all elements other than theactuating sequence (here, encoding lacZ) are flanked upstream anddownstream by Rox sites. Upon exposure to Dre recombinase, only theactuating sequence is present. Excision of the Roxed sequences can beconfirmed by loss of the DSC (here, containing neo^(r)), and/or loss ofthe COIN, and/or loss of the NSI. The result is a true null allele thatlacks the DSC, NSI, and COIN.

An MFA as illustrated in FIG. 2 and as exemplified at the top of FIG. 3can be used to create a conditional allele. A conditional allele can begenerated by selecting the appropriate recombinase with which to exposethe allele in the first instance. The appropriate recombinase in thisembodiment is a recombinase that inverts the NSI back to the sensestrand and leaves the COIN in the antisense orientation. This can beachieved, e.g., by exposing the MFA to a recombinase that recognizesR1/R1′ and also R3/R3′ (e.g., a Flp recombinase where R1/R1′ and R3/R3′are selected from FRT and FRT3 sites; see FIG. 5 for a particularembodiment). Briefly, once an MFA is placed at a desired location in agenome, it can be used to generate a conditional allele, wherein theinverted NSI of FIG. 2 and the top of FIG. 3 is disposed in anorientation for transcription of the target gene, while leaving the COINin antisense orientation and deleting the actuating sequence and DSC. Anexample of this embodiment is shown in FIG. 5, where an actuatingsequence that contains a lacZ and a DSC containing neo^(r) are removedby first exposing the allele to Flp recombinase, causing an inversion ofelements directed by FRT3 sites, followed by Flp-mediated deletiondirected by FRT sites. The resulting allele presents the NSI in anorientation for transcription, but leaves the COIN in the antisenseorientation.

As shown in FIG. 6, the same conditional allele can be achieved whetherFlp-mediated inversion occurs first via FRT sites (as in FIG. 5) or FRT3sites (as in FIG. 6).

In the embodiment that generates a conditional allele, recombinase sitesremaining in the allele are selected such that treatment with one ormore suitable recombinases results in subsequent deletion of the NSI (orre-inversion of the NSI) and inversion of the COIN, such that the alleleresults in a null allele with respect to the NSI but also places theCOIN in orientation for transcription. An example of this is shown usingloxP and lox2372 sites, which each independently direct Cre-mediatedrecombination. Although Cre-reactive sites are used, any suitable sitescan be used instead of Cre sites.

As shown in FIG. 7, the NSI in sense orientation (i.e., in position fortranscription and translation) is disposed 3′ with respect to a firstlox2372 site. Following the NSI is a first loxP site in the sameorientation as the first lox2372 site, and an inverted (i.e., antisense)COIN is placed downstream of the first loxP site, and the inverted COINdisposed upstream of a second lox2372 site in opposite orientation withrespect to the first lox2372 site. Disposed downstream of the secondlox2372 site is a second loxP site disposed in an opposite orientationwith respect to the first loxP site. This arrangement allows, upontreatment with Cre, inversion via either lox site followed by deletionvia either lox site (see FIG. 7). The resulting allele contains a COINin sense orientation, i.e., in position for transcription andtranslation.

In an alternative arrangement (see FIG. 8), a loxP site is placed 5′with respect to the NSI (instead of disposed between the NSI and theCOIN), such that exposure to Cre results in inversion of the NSI toantisense orientation and the COIN to sense orientation.

The MFA approach provides options for many embodiments. In a specificembodiment, upon exposure to the first recombinase, the arrangement ofelements and recombinase sites are as shown in the bottom construct ofFIG. 5 or FIG. 6, wherein the FRT3 site as shown is site R1 that has nocognate site in the resulting allele, the FRT site as shown is arecombinase site R3 that has no cognate site in the resulting allele,the left-most lox2372 site is site R5 that is paired with a cognaterecombinase site R5′ occupying the right-most lox2372 site as shown, theleft-most loxP site as shown is site R4 that is paired with a cognaterecombinase site R4′ provided by the right-most loxP site as shown, andthe Rox site as shown is site R2 that has no cognate recombinase site inthe resulting allele.

In a specific embodiment, upon exposure to the second recombinase, thearrangement of elements and recombinase sites of the resulting alleleare as shown in the bottom construct of FIG. 7, wherein the FRT3 siteshown is site R1 that has no cognate site in the resulting allele, thelox2372 site is site R5 that is not paired with a cognate recombinasesite in the resulting allele, the FRT site shown is site R3 that is notpaired with a cognate recombinase site in the resulting allele, the loxPsite as shown is site R4 that is not paired with a cognate recombinasesite in the resulting allele, and the Rox site as shown is site R2′ thatis not paired with a cognate recombinase site in the resulting allele.

In a specific embodiment, the resulting allele allows expression of theCOIN following exposure to the second recombinase. In a specificembodiment, the COIN is a reporter or a DSC.

In one aspect, an MFA is provided that comprises a COIN, an NSI, a DSC,a reporter, and recombinase sites that are arranged such that action byone recombinase will excise the COIN, the NSI, and the DSC but not thereporter (FIG. 11B), whereas action with a different recombinase willgenerate an allele that lacks the DSC but that places the NSI in senseorientation while maintaining the COIN in antisense orientation (FIG.11C). This resulting allele has recombinase sites arranged such thataction by a further recombinase will excise the NSI and place the COINin sense orientation (FIG. 11D). Thus, in the embodiments discussed,this MFA will allow selection of a true knockout with a reporterfunction and removal of the DSC, or placement of an NSI, whereinsubsequent removal of the NSI is confirmed by concomitant placement of aCOIN in sense orientation. A schematic of some overlapping recombinaseunits are shown in FIG. 11A for such an allele, with like recombinaseunits represented by like dashed shapes.

In one aspect, an MFA is provided that comprises a COIN, an NSI, a DSC,a reporter, and recombinase sites that are arranged such that action byone recombinase will excise the NSI and DSC but maintain the orientationof the reporter and COIN (FIG. 12B), whereas action with a differentrecombinase will generate an allele that lacks the DSC and reporter butthat places the NSI in sense orientation while maintaining the COIN inantisense orientation (FIG. 12C). This resulting allele has recombinasesites arranged such that action by a further recombinase will excise theNSI and place the COIN in sense orientation (FIG. 12D). Thus, in theembodiments discussed, this MFA will allow selection of a true knockoutwith a reporter function and removal of the DSC; or placement of an NSI,wherein subsequent removal of the NSI is confirmed by concomitantplacement of a COIN in sense orientation. A schematic of someoverlapping recombinase units are shown in FIG. 12A for such an allele,with like recombinase units represented by like dashed shapes.

In one aspect, an MFA is provided that comprises a COIN, an NSI, a DSC,a reporter, and recombinase sites that are arranged such that action byone recombinase will excise the reporter and DSC and place the NSI insense orientation (FIG. 13B). This resulting allele has recombinasesites arranged such that action by a further recombinase will place theNSI in antisense orientation while placing the COIN in sense orientation(FIG. 13C). Thus, in the embodiments discussed, this MFA will allowcreation of a conditional allele from an MFA.

In one aspect, an MFA is provided that comprises a COIN, and NSI, a DSC,a reporter, and a different array of recombinase sites that are arrangedsuch that action by a selected recombinase will excise the reporter andthe DSC and place the NSI in sense orientation (FIG. 14B). Thisresulting allele has recombinase sites arranged such that action by afurther recombinase will place the NSI in antisense orientation whileplacing the COIN in sense orientation (FIG. 14C). Thus, this MFA willalso allow creation of a conditional allele from an MFA.

In one aspect, an MFA is provided that comprises an NSI, a DSC, areporter, a COIN, and recombinase sites that are arranged such thataction by a selected recombinase will excise the reporter and the DSCand place the NSI in sense orientation while maintaining the COIN inantisense orientation (FIG. 15B). This resulting allele has recombinasesites arranged such that action by a further recombinase will place theNSI in antisense orientation while placing the COIN in sense orientation(FIG. 15C). Thus, this MFA will also allow creation of a conditionalallele from an MFA.

EXAMPLES Example 1 Hprt1 MFA

Hprt1 is a gene that is X-linked in mice, and Hprt1-null ES cells areresistant to the nucleobase analog 6-thioguanine (6-TG). This propertyprovides an easy and robust phenotypic test, as cells that are wild typefor Hprt1 die in the presence of 6-TG, whereas cells that are null forHprt1 survive. Additionally, if one targets ES cells that are derivedfrom male blastocysts (as is typically the case, and is also the casefor the majority of ES cell lines currently in use for targeting), thenonly one round of targeting is needed to generate Hprt1^(MFA)/Y EScells. In order to generate Hprt1^(MFA)/Y ES cells, an MFA in atargeting vector, according to the allele shown in FIG. 5 (top), isprepared by standard genetic engineering methodology and bacterialhomologous recombination according to the VELOCIGENE® method describedin U.S. Pat. No. 6,586,251 and in Valenzuela et al. (2003)High-throughput engineering of the mouse genome coupled withhigh-resolution expression analysis, Nature Biotech. 21(6):652-659 (thepatent and article are hereby incorporated by reference). TheHprt1^(MFA) allele is designed around exon 3, defining exon 3 and theconserved intronic sequence directly 5′ and 3′ of it as the NSI (FIG.9). The reason for this choice lies in that exon 3 begins in frame 2(f2) and ends in frame 0 (f0); by extension, the preceding exon exon 2)ends in frame 2 (f2), and the following exon (i.e., exon 4) begins inframe 0 (f0). This means that if this NSI is inverted into the antisenseorientation, then exon 2 is rendered out of frame with respect to exon4, because exon 2 ends in f2 and exon 4 starts in f0. In this manner, ifin the Hprt1^(MFA) allele there is any transcription past the actuatingsequence—SA-/acZ-polyA (FIG. 10)—and there is also splicing that removesthe actuating sequence from the final mRNA, that mRNA will not compriseexon 3 and will encode a nonsense sequence, effectively giving rise toan Hprt1-null mRNA and phenotype. Conversely, for the Hprt1^(COIN-INV)allele (generated by treatment of Hprt1^(MFA) with FLP or variants ofFLP to first generate the Hprt1^(COIN) allele, then by treatment withCre to generate Hprt1^(COIN-INV)) if there is transcription past theSA-eGFP-polyA of the COIN element (FIG. 10), and there is also splicingthat removes the SA-eGFP-polyA sequence from the final mRNA, that mRNAwill not comprise exon 3 and therefore will encode a nonsense sequence,effectively giving rise to an Hprt1-null mRNA and phenotype.

The antisense-oriented NSI is exon 3 and surrounding evolutionarilyconserved intronic sequence of Hprt1 (FIG. 9), and theantisense-oriented COIN is a SA-eGFP-polyA. The targeting vector has amouse homology arm upstream of the first FRT3 site and downstream of thesecond Rox site that direct the targeting into the Hprt1 locus such thatit is replaced by its MFA version, whereby (a) a SA-LacZ-polyA elementin the sense orientation with respect to the direction of transcriptionof Hprt1, followed by a DSC in the antisense orientation with respect tothe direction of transcription of Hprt1, both preceding exon 3 of Hprt1,(b) exon 3 is placed into the antisense orientation with respect to thedirection of transcription of Hprt1, and (c) a COIN element is placed inthe antisense orientation with respect to the direction of transcriptionof Hprt1 downstream of the exon 3, and where these different elementsare flanked by site-specific recombinase recognition sites, togetherarranged in recombinable units as detailed in FIG. 3 and FIG. 10, withSA-LacZ-polyA being the actuating sequence, and exon 3 plus flankingintronic sequences of Hprt1 being the NSI.

The targeting vector is prepared and electroporated into ES cellsaccording to the VELOCIGENE® method described in U.S. Pat. No. 6,586,251and in Valenzuela et al. (2003) High-throughput engineering of the mousegenome coupled with high-resolution expression analysis, Nature Biotech.21(6):652-659 (the patent and article are hereby incorporated byreference). The resulting ES cells bear the MFA allele of Hprt1 in placeof the wild type version of Hprt1. Prior to any further modification theHprt1^(MFA)/Y ES cells are resistant to treatment with 6-TG (becausethey are effectively null for Hprt1), demonstrating the usefulness ofthe MFA method to generate a true knockout-first allele. After treatmentwith Dre, this property is preserved, while the genotype of the cells isconverted to Hprt1^(SA-LacZ-polyA)/Y. Although for the Hprt1 locus, thismodification may neither alter the expression level of the reporter(LacZ) nor have any phenotypic consequences (alter resistance to 6-TG),this may not be the case for other loci. After treatment with FLP or FLPvariants, in a step that is effectively equivalent to a complementationtest, the Hprt1^(MFA)/Y ES cells are converted to Hprt1^(COIN)/Y EScells which are effectively wild type and hence sensitive to 6-TG. Inaddition, this operation restores expression of the Hprt1 message backto its wild-type identity. After treatment with Cre, the Hprt1^(COIN)/YES cells are converted to Hprt1^(COIN-INV)/Y ES cells which areeffectively null for Hprt1 and hence resistant to 6-TG. In addition,this operation results in abrogation of expression of the wild-typemessage of Hprt1 message, and its concomitant replacement with a hybridmessage composed of the first exon of Hprt1 and eGFP (encoded by theCOIN element), thereby generating an allele that expresses eGFP in placeof Hprt1. This new property, expression of eGFP, can be optionally usedto score for inversion of the COIN element to the sense strand, and hasfurther utility in enabling the isolation of cells where this event hastaken place from a cell population where both types of cells(Hprt1^(COIN)/Y ES cells, and Hprt1^(COIN-INV)/Y ES cells) exist.Therefore, not only is the COIN allele converted into a null, but theevent is also marked by a new, easily measurable and useful event.

Example 2 Hprt1 MFA Results

An MFA having a LacZ reporter (SA(adml)-gtx-LacZ-pA) in senseorientation, a neomycin DSC (Neo), an NSI in antisense orientation thatencompasses a critical exon (e_(c)) for Hprt1 (exon 3) and flankingevolutionarily conserved intronic sequences, and a COIN(Gtx-SA-HA-myc3-TM-T2A-GFP-pA) was constructed with an arrangement ofrecombinase sites as shown in FIG. 16A. The MFA was electroporated intoF1H4 ES cells and were selected for resistance to G418. Subsequently,G418-resistant colonies were genotyped to determine targeting. Fivetargeted clones (Hprt1^(MFA)/Y) were obtained from a total of 96colonies screened. All five of these clones were found to survive andpropagate when cultured in standard ES cell media supplemented with 10μM 6-TG (which is the standard 6-TG survival assay utilized), as wouldbe expected for cells that are Hprt1-null (Doetschman, T. et al. (1987)Targeted correction of mutant HPRT gene in mouse embryonic stem cells,Nature 330:576-578). In contrast, the parental cell line, F1H4, as wellas any of the non-targeted clones that were tested, failed to grow inthe presence of 6-TG. These results are in agreement with what has beenreported previously (Doetschman et al. (1987)).

Upon treatment with recombinase FLPo (Raymond, C. S. and Soriano, P.(2007) High-efficiency FLP and PhiC31 site-specific recombination inmammalian cells, PLoS ONE 2:e162), the Hprt1^(MFA) allele is convertedto the Hprt1^(COIN) allele (FIG. 16B), giving rise to Hprt1^(MFA)/Y EScells. This operation results in removal of the LacZ reporter, the DSC,as well as in re-inversion of the NSI into the sense strand. Therefore,the resulting allele (Hprt1^(COIN)) is functionally wild type, as thewild type Hprt1 mRNA is encoded and expressed.

On further treatment with recombinase Cre (Sauer, B. and Henderson, N.(1988) Site-specific DNA recombination in mammalian cells by the Crerecombinase of bacteriophage P1, Proc. Natl. Acad. Sci. USA85:5166-5170), the Hprt1^(COIN) allele is converted to theHprt1^(COIN-INV) allele (FIG. 16C), giving rise to Hprt1^(COIN-INV)/Y EScells. This allele (Hprt1^(COIN-IN V)) is functionally null, as theHprt1 mRNA is replaced by one encoding eGFP (and is also lacking theNSI—i.e., Hprt1's exon 3 and flanking intronic sequences as defined atthe design stage).

Cells bearing the MFA (Hprt1^(MFA)/Y) were tested for resistance to thenucleotide analog 6-TG, and were compared with wild-type cells (FIG.17). The Hprt1^(MFA)/Y ES cells survived whereas the Hprt1⁺/Y ES cellsdied, indicating that the Hprt1^(MFA)/Y are functionally Hprt1-null.Hprt1^(MFA)/Y ES cells were then treated with FLPo, to test if theHprt1^(MFA) allele would be converted to the Hprt1^(COIN) allele. Theresulting Hprt1^(COIN)/Y ES cells are expected to be phenotypically wildtype, as Hprt1 expression is restored. This was shown to indeed be thecase, as Hprt1^(COIN)/Y ES cells die when cultured in the presence 6-TG,just like their wild type (Hprt1⁺/Y) counterparts. Finally, theHprt1^(COIN)/Y ES cells were treated with Cre to generateHprt1^(COIN-INV)/Y ES cells, which are predicted to be null for Hprt1 asthe COIN module is activated while simultaneously deleting Hprt1's exon3 (FIG. 16, Panel C). When cultured in the presence of 6-TG, theHprt1^(COIN-INV)/Y ES cells survived and proliferated, confirming thatthey are functionally null for Hprt1, as intended by the MFA design andapplication.

The phenotypic results obtained above where further confirmed at theprotein level, by performing Western blots on protein preparations of EScells belonging to each genotypic class: wild-type (Hprt1⁺/Y),Hprt1^(MFA)/Y (MFA), Hprt1^(COIN)/Y (MFA+FLPo), and Hprt1^(COIN-INV)/Y(MFA+FLPo+Cre). These protein preparations were examined for reporterand NSI (i.e., Hprt1) expression. Hprt1^(MFA)/Y ES cells lack Hprt1protein, but express the reporter (LacZ). In Hprt1^(COIN)/Y ES,expression of Hprt1 is restored to wild type levels, reflecting theplacement of the NSI (exon 3 of Hprt1) back into the sense orientation,and did not show reporter (LacZ) protein, confirming reporter excisionby FLPo. This established that the Hprt1^(MFA) allele is indeed null,and can be converted to a functional wild type allele after removal ofthe reporter and DSC, and concomitant re-inversion of the NSI into thesense strand (an operation experimentally accomplished by FLPo). Thefact that at the level of Hprt1 protein expression the Hprt1^(COIN)allele is identical to wild type (Hprt1⁺), further demonstrates therobustness of this method to generate a true conditional-null andperform the equivalent of a complementation assay in one,recombinase-mediated, post-targeting step. Finally, theHprt1^(COIN-INV)/Y ES cells lack Hprt1 protein, effectively confirmingthe phenotypic observations made using the 6-TG resistance assay. Thisfurther confirms that the COIN-based conditional-null allele(Hprt1^(COIN)) functions as intended.

We claim:
 1. A nucleic acid construct, comprising: (a) targeting armsfor directing the nucleic acid construct to a target gene of a nucleicacid of a cell; (b) an actuating sequence in sense orientation withrespect to transcription of the target gene, and a drug selectioncassette (DSC) in sense or antisense orientation; (b) in antisenseorientation a nucleotide sequence of interest (NSI) and a COIN; and, (c)recombinable units that recombine upon exposure to a first recombinaseto form a conditional allele that (i) lacks the actuating sequence andthe DSC, and, (ii) contains the NSI in sense orientation and the COIN inantisense orientation; wherein the recombinable units comprise two firstpairs of cognate recombinase recognition sites recognized by the firstrecombinase, two second pairs of cognate recombinase recognition sitesrecognized by a second recombinase, and one third pair of cognaterecombinase recognitions sites recognized by a third recombinase;wherein the first pairs, the second pairs, and the third pairs ofrecombinase recognition sites are recognized by different recombinases.2. The nucleic acid construct of claim 1, wherein the conditional allelewhen further exposed to a second site-specific recombinase, recombinesto form an allele lacking the NSI and having the COIN in senseorientation.
 3. A method for modifying a non-human cell, comprising:targeting a nucleotide sequence of interest in a cell with a nucleicacid construct of claim 1 to form a targeted cell, exposing the targetedcell to the first recombinase to form the conditional allele, andexposing the conditional allele to a second recombinase that excises theNSI and places the COIN in sense orientation.
 4. The method of claim 3,wherein the NSI in the cell is an exon or a nucleotide sequence that isassociated with a phenotype.
 5. The method of claim 4, wherein thenucleotide sequence of interest in the cell is replaced by the nucleicacid construct of claim
 1. 6. The method of claim 5, wherein thephenotype is determined a first time after forming the targeted cell butbefore exposure to the first recombinase.
 7. The method of claim 6,wherein the phenotype is determined a second time following exposure tothe first recombinase.
 8. A nucleic acid construct, comprising: a drugselection cassette (DSC), a reporter, a COIN, a nucleotide sequence ofinterest (NSI), and five pairs of recombinase sites arranged among thereporter, the DSC, the COIN, and the NSI; wherein no pair of recombinasesites is identical to any other pair, and wherein a first two pairs ofrecombinase sites are recognized by the same first recombinase, a secondtwo pairs of recombinase sites are recognized by the same secondrecombinase, and a fifth pair of recombinase sites are recognized by athird recombinase; wherein the first, second, and third recombinases arenot the same, and wherein, with respect to direction of transcription,the reporter is in sense orientation, the NSI is in antisenseorientation, the COIN is in antisense orientation, and the DSC is insense or antisense orientation; and, wherein the first two pairs and thesecond two pairs of recombinase sites are arranged such that uponexposure to the first recombinase, a modified allele forms wherein thefirst two pairs of recombinase sites direct excision of the reporter,excision of the DSC, inversion of the NSI to sense orientation, and theCOIN is maintained in antisense orientation, and either (a) the secondtwo pairs of recombinase sites are arranged such that upon exposure tothe second recombinase the NSI is excised and the COIN is placed insense orientation; or (b) the second two pairs of recombinase sites arearranged such that upon exposure to the second recombinase the NSI isplaced in antisense orientation and the COIN is placed in senseorientation.
 9. A nucleic acid construct according to claim 8, whereinthe fifth pair of recombinase sites are arranged such that upon exposureto the third recombinase and in the absence of exposure to the first orthe second recombinase, the fifth recombinase excises that COIN, theNSI, and the DSC, and maintains the reporter in the sense orientation.10. A nucleic acid construct according to claim 8, wherein the nucleicacid construct is inserted into a gene, and with respect to thedirection of transcription of the gene, from 5′ to 3′, the constructcomprises a reporter in sense orientation, a DSC in sense or antisenseorientation, an NSI in antisense orientation, and a COIN in antisenseorientation.
 11. A nucleic acid construct according to claim 10, whereinthe DSC is in sense orientation.
 12. A nucleic acid construct accordingto claim 8, wherein the nucleic acid construct is inserted into a gene,and with respect to the direction of transcription of the gene, from 5′to 3′, the construct comprises a COIN in antisense orientation, an NSIin antisense orientation, a DSC in sense or antisense orientation, and areporter in sense orientation.
 13. A nucleic acid construct according toclaim 12, wherein the DSC is in sense orientation.
 14. A nucleic acidconstruct according to claim 8, wherein the nucleic acid construct isinserted into a gene, and with respect to the direction of transcriptionof the gene, from 5′ to 3′, the construct comprises an NSI in antisenseorientation, a DSC in sense or antisense orientation, a reporter insense orientation, and a COIN in antisense orientation.
 15. A nucleicacid construct according to claim 14, wherein the DSC is in senseorientation.
 16. A nucleic acid construct according to claim 8, whereinthe nucleic acid construct is inserted into a gene, and with respect tothe direction of transcription of the gene, from 5′ to 3′, the constructcomprises a reporter in sense orientation, a DSC in sense or antisenseorientation, an NSI in antisense orientation, and a COIN in antisenseorientation.
 17. A nucleic acid construct according to claim 16, whereinthe DSC is in sense orientation.
 18. A nucleic acid construct accordingto claim 8, wherein the nucleic acid construct is inserted into a gene,and with respect to the direction of transcription of the gene, from 5′to 3′, the construct comprises a COIN in antisense orientation, an NSIin antisense orientation, a DSC in sense or antisense orientation, and areporter in sense orientation.
 19. A nucleic acid construct according toclaim 18, wherein the DSC is in sense orientation.
 20. A nucleic acidconstruct according to claim 8, wherein the nucleic acid construct isinserted into a gene, and with respect to the direction of transcriptionof the gene, from 5′ to 3′, the construct comprises an NSI in antisenseorientation, a DSC in sense or antisense orientation, a reporter insense orientation, and a COIN in antisense orientation.